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The nucleic acid protein interaction database (npidb) provides an access to information about all available structures of dna protein and rna protein complexes . Almost 4000 structures of such complexes are now available in the protein data bank [pdb, (1)]. The npidb automatically extracts data from the pdb, then the data are presented through a user - friendly interface . A number of tools for the analysis of interactions between protein and nucleic acid molecules are offered . Among the tools are the original program clud for analysis of hydrophobic clusters on interfaces (2,3), the program hb - angles for detecting potential hydrogen bonds and water bridges and visualization of structures with jmol (4). The database includes a classification of nucleoprotein complexes based on protein domain families according to pfam (5) and structural classification of proteins [scop (6)]. There are a number of online databases providing information on dna protein or rna protein complexes . The 3d - footprint (7), http://floresta.eead.csic.es/3dfootprint, contains information on dna protein complexes . Its functionality partially overlaps the functionality of the npidb . Among the differences, we would like to mention the following . Inter - molecular interactions in the 3d - footprint are visualized as a number of schematic pictures, while the npidb provides visualization of interactions with jmol . Parts of biological units that are not presented explicitly in a pdb entry and should therefore be reconstructed by symmetry are not taken into account in the 3d - footprint . On the other hand, the 3d - footprint contains comparative information on dna protein interfaces, providing, for example, a dendrogram of similar interfaces, position weight matrices illustrating the binding specificities and other tools . It also allows searching the dna sequence motifs recognized by dna - binding proteins . The protein - dna interface database [pdidb (8)], http://melolab.org/pdidb/web/content/home, contains descriptions of dna protein interfaces for 922 x - ray dna protein complexes with resolution better than 2.5 (the npidb contains 1193 such complexes). These complexes are partitioned into several classes according to function of proteins, the classes are further divided into types . For each complex, information on different types of contacts can be visualized or downloaded as a text table . The protein - rna interface database [pridb (9)], http://bindr.gdcb.iastate.edu/pridb, contains structural information on rna protein complexes . Biological interaction database for protein - nucleic acid [bipa (10)], http://mordred.bioc.cam.ac.uk/bipa/, contains both dna protein and rna protein complexes . At the moment, the total number of complexes in the bipa is 2570 (the npidb contains 3846 complexes). Information on inter - molecular contacts is presented in graphical mode only, not in a parsable format . Web server of protein - dna complex structure analyzer [webpda (11)], http://bioinfozen.uncc.edu/webpda/, contains data on contacts between dna and protein molecules in dna protein complexes . Contacts are presented both via an online jmol applet and in a parsable text format . The protein - dna recognition database (http://gibk26.bio.kyutech.ac.jp/jouhou/3dinsight/recognition.html) consists of three main parts: the protein - nucleic acid complex database (pronuc), the database of base - amino acid interactions (baint) and the thermodynamic database for protein - nucleic acid interactions [pronit, (12)]. The pronuc allows searching over protein dna complexes by pdb code, name of protein, type of dna - recognizing motif and some other features . The baint allows finding information on interaction of amino acids of a certain type (e.g. Lysine) with dna nucleotides of a certain type . The nucleic acid database [ndb (13)], http://ndbserver.rutgers.edu/, is focused on structure of nucleic acid molecules, not on their interaction with proteins . A number of new features were added, among them: (i) a new search engine; (ii) improved storage and visualization of different kinds of nucleic acid protein interactions; (iii) detection of pfam domains in new entries with hmm profiles; (iv) classification of dna protein interaction modes; and (v) information on conserved water molecules at protein nucleic acid interfaces . Structures of protein nucleic acid complexes are extracted from the pdb as files in pdb format representing both pdb entries (asymmetric units) and biological units . For each biological unit of x - ray files and for first models of nuclear magnetic resonance files, the interactions of three kinds are considered, namely, hydrogen bonds, hydrophobic interactions and water bridges (water molecules that form hydrogen bonds both with protein and dna). For each protein chain, its secondary structure is detected by stride (18) and stored in the database . Each protein chain of each structure is analysed to identify pfam and scop domains (5,6). Pfam domains are determined by means of hidden markov model (hmm) profiles of the pfam database . The profiles are downloaded from the pfam ftp site (ftp://ftp.sanger.ac.uk/pub/databases/pfam/current_release). To search pfam domains in the sequences, hmmer (19) software downloaded from the web site (http://hmmer.janelia.org/) and the pfamscan script (ftp://ftp.sanger.ac.uk/pub/databases/pfam/tools/pfamscan.tar.gz) information on scop domains is extracted by perl scripts from scop parsable files (http://scop.mrc-lmb.cam.ac.uk/scop/parse/index.html), release 1.75 . For each (pfam or scop) domain, a structure file in pdb format is created . This file contains description of the domain itself and of segments of nucleic acid chains that are in contact with the domain . Sets of representatives of pfam and scop families (one complex for each family containing at least one domain with a known x - ray structure) are created and stored . These representatives are chosen from the complexes with best resolution among all complexes representing each particular family . Namely, there are 1847 scop domains in contact with double - stranded dna with at least 10 complementary base pairs . For each of these 110 families, all their representatives were extracted from the pdb, including those that were solved in the absence of dna . The service pdbefold [protein structure comparison service fold at european bioinformatics institute, http://www.ebi.ac.uk/msd-srv/ssm, (20)] was used . In a number of cases, some families were divided into subfamilies; this was done if it was impossible to superimpose the entire family with an appropriate quality . For each superimposed set of protein chains, the sequence alignment generated by pdbefold the superimposed families are partitioned into interaction classes according to the modes of their dna the definition of each interaction class takes into account, first, the part of dna involved into the interaction and specificity of contacts, namely, specific contacts through major groove, specific contacts through minor groove or non - specific contacts, and, second, the contacting elements of protein secondary structure, namely, alpha helices, beta strands and other chain segments called loops. To determine the interaction class of a family, we determine, first, the main dna groove (major or minor) involved in specific interaction, and, second, the secondary structure elements involved in specific interaction with the main groove . At this step, a human judgement is essential in a number of cases because family representatives may vary in minor additional interactions . Thus, we have the following groups of families: (i) the main groove is the major one, and the elements are helices only; (ii) the main groove is the major one, and the elements are helices and loops and so on . There are two grooves and seven possible combinations of three secondary structure elements; hence, there are 14 possible groups . At the moment, each of these 14 groups is divided into two classes, depending on presence or absence of small specific interaction via the other, not the main one, dna groove . Additionally, there are two more interaction classes: one, with almost equal role of both grooves in specific interaction, and, second, with non - specific contacts only . Altogether, there are 30 possible classes, but only 20 classes are really represented by dna - recognizing protein domains . Among these 20 classes, a total of 13 families belong to the class main specific contacts via the dna major groove, some specific contacts via the dna minor groove, contacting protein elements: helices and loops, 12 families belong to the class main specific contacts via the dna major groove, some specific contacts via the dna minor groove, contacting protein elements: helices, 11 families belong to the class specific contacts via the dna major groove only, contacting protein elements: helices. Some classes contain only one family each, for example, the class specific contacts via the dna major groove and the dna minor groove in almost equal level contains only the family ten classes are not presented at all, among them are eight classes where beta strands, but not loops, are involved, and also the classes where specific contacts are with the dna minor groove only and among the contacting elements are helices and loops together . The list of interaction classes is available on the npidb web site . For each interaction class, a set of representatives of families of the type (the subset of best resolution representatives of all scop families) is available . For the 73 superimposed families of dna - binding protein domains, information on conserved positions of water molecules at dna protein interface is computed with the wlake program (21) and stored in the npidb . Information about water molecules in pdb files is usually less precise and less confident than analogous information about atoms of protein or dna . However, if a water molecule is presented in several structures of a same complex or a number of closely related complexes in approximately same place, then this molecule can be considered as confident and important . That is why we revealed so - called conserved water bridges that are water molecules at dna protein interfaces in similar complexes that approximately coincide after superposition of macromolecules . A detailed description of the procedure that reveals conserved water bridges is given in (21). In brief, the procedure is as follows . As an input, the procedure uses a set of superimposed structures of protein domains from the given family . Thus, a set of similar structures placed in a common coordinate space is analysed . A conserved water bridge is a set of water molecules from different structures such that: (i) the distance between any two water oxygen atoms in the common space is <1.5; (ii) each water molecule is closer than 5 to a dna atom from the same or other structure; and (iii) each water molecule is closer than 5 to a protein atom from the same or other structure . Lists of conserved water bridges and their visualization are available at pages of correspondent scop families . For a number of pfam families (hom_end, ets and some other), a manually created description of their interaction with dna is available . All structural files of the npidb are downloadable, as well as tables of structures, domain families and interactions . A perl script identifies chains of dna, rna and protein directly in the coordinate (atom and hetatm) section of a pdb file . If a pdb file contains at least one protein chain and at least one nucleic acid chain, then it is selected for incorporating into the npidb . The search engine allows searching entries according to the following criteria: pdb i d, year of deposition in pdb, keywords (header, title and organism fields of the pdb), experimental method, resolution and type of the nucleic acid (dna, rna or hybrid). For storing derived data, a relational database under mysql the data include headers, titles and creation dates of pdb entries, types of chains, secondary structure of protein chains, data on protein nucleic interaction and coordinates (start end) of scop and pfam domains in protein chains . The main part of information also, the database contains sequence files in fasta format and output files of stride and the programs computing interactions . Perl and python scripts are used for regular update of the content of the relational database and collection of data files . Other scripts generate dynamic web pages using data both from the relational database and from the data files . Hydrogen bonds and water bridges calculations are based on a statistical potential derived from analysis of all available 3d structures of macromolecules . The potential for a hydrogen bond is the product of three factors: the first factor depends on the distance between the centres of the donor and acceptor atoms; the second factor depends on the angle formed by the donor, the acceptor and the covalently bound neighbour of the donor; the third factor analogously depends on the angle formed by the donor, the acceptor and the covalently bound neighbour of the acceptor . A hydrogen bond is detected if the potential is> 0.1, and the distance between the centres of the donor and acceptor atoms is <3.7 . A water bridge is detected if there exists a water molecule that forms hydrogen bonds both with the protein molecule and the nucleic acid molecule . The program hb - angles performing the computations is written in c. for interactive visualization of individual hydrogen bonds and water bridges, javascript is used . The program clud (2,3) for detecting hydrophobic clusters in macromolecular structures is integrated into the npidb . The main principle of the program is obtaining clusters of hydrophobic atomic groups (such as methyl groups of thymine or threonine) that fill some volume and thus displace the solvent . Thus, clud outputs hydrophobic clusters rather than pairwise residue - to - residue hydrophobic interactions . The clusters may include atoms not only from entirely hydrophobic residues (e.g. Leucine) but also from hydrophobic parts of side chains of lysine, glutamine and so on . The main optional parameter of clud is the threshold for the distance between centres of hydrophobic groups (that are carbon and sulphur atoms); at the moment, the threshold is set to 5.4, which is the theoretically maximal distance allowing two non - polar groups to displace a water molecule (it is the sum of two van der waals radii of methyl groups and the diameter of a water molecule). There are four entry points to the content, namely, the list of all complexes, the list of pfam families, the list of scop families and the list of interaction classes of dna - recognizing scop domains . Pdb id, date (the date of deposition to the pdb), pdb header, experimental method, resolution and kind (type of the nucleic acid: dna, rna or hybrid). The table can be sorted according to any column with a click on the column header . The page of each entry contains the following sections (figure 1): general information (pdb i d, title, pdb header, date, experimental method, resolution, type of nucleic acid and organism);quick links to other databases;composition of the pdb entry and biounits: chains of the protein and the nucleic acid and models;lists of pfam and scop domains presented in the entry;sequences of the chains with additional information: secondary structure and contacts with the nucleic acid for the protein chains and contacts with the protein for the nucleic acid chains . For each biological unit, there are pages with information on interaction between molecules of the nucleic acid and the protein . Three types of interaction are presented, namely, hydrogen bonds (figure 2), water bridges and hydrophobic interactions . General information (pdb i d, title, pdb header, date, experimental method, resolution, type of nucleic acid and organism); quick links to other databases; composition of the pdb entry and biounits: chains of the protein and the nucleic acid and models; lists of pfam and scop domains presented in the entry; sequences of the chains with additional information: secondary structure and contacts with the nucleic acid for the protein chains and contacts with the protein for the nucleic acid chains . A screenshot of the page of the complex 3exj . A screenshot of the page that shows hydrogen bonds in the complex 1a1k . Pfam id, domain (short description of the family from pfam), number of entries (number of complexes with representatives of the family), number of domains (number of domains from the family in all complexes), number of bound domains (number of domains bound to dna or rna). The page of each family contains the list of entries (with hyperlinks to entries pages). For a number of families (for example, for the list of scop families is designed as a tree of scop classes, folds, superfamilies and families . A hyperlink from each family name leads to a page containing a table analogous to a table of a pfam family, and, for a number of families of dna - recognizing domains contacting with a long (> 10 bp) double - stranded dna, a description of the family . The description includes a structural superposition of all representatives of the family in the pdb, the corresponding multiple amino acid sequence alignment and an information on conserved water bridges on the protein dna interface . The list of interaction classes of dna - recognizing scop domains contains hyperlinks to lists of scop families whose representatives demonstrate the certain mode of dna protein interaction . Russian foundation of basic research [grant 10 - 07 - 00685-a and joint grant with german research foundation 11 - 04 - 91340]; ministry of education and science of the russian federation [state contract no . 07.514.11.4006].
Age - related macular degeneration (amd) is the leading cause of irreversible, severe visual loss in people aged 55 and older in the developed world (congdon et al 2004) and it is estimated that more than 500 000 people worldwide lose their sight annually from the disease (eyetech pharmaceuticals, inc . 2005). The neovascular (wet) form of the disease accounts for only 10% of the total incidence of the disease, but is responsible for 90% of the severe visual loss associated with the disease (ferris et al 1984). Within the next 5 years, it is expected to affect almost 1 million people in the usa, posing a severe health issue (bressler et al 2003) and having a major impact on the quality of life for the elderly, due to difficulties in performing routine tasks (dong et al 2004). Wet amd is characterized by choroidal neovascularization (cnv) that penetrates bruch s membrane and invades the subretinal space, often leading to exudation and hemorrhage (green 1999; pauleikhoff 2005). If left untreated, damage results to the photoreceptors leading to loss of central vision and eventually the vessels are largely replaced by a fibrovascular scar (green 1999). The visual prognosis is variable, based on lesion location, composition, and size (pauleikhoff 2005). Other factors are involved, but it is clear that vascular endothelial growth factor (vegf) is a key molecule in the development of cnv . Vegf is regulated by hypoxia and it promotes angiogenesis and vasopermeability, which are characteristic of the disorder (damore 1994; green 1999; pauleikhoff 2005). Vegf and its mrna are upregulated in cnv associated with amd (kvanta et al 1996; lopez et al 1996; wells et al 1996) and in experimental models of cnv (ishibashi et al 1997; yi et al 1997). Vegf is critical for experimental cnv to develop (vinores et al 2006) and exposure of choroidal vessels to vegf results in cnv formation (schwesinger et al 2001). Collectively, these data provide a strong rationale for targeting vegf in the treatment of wet amd . To utilize this strategy, pegaptanib (macugen), a 28-base ribonucleic acid aptamer, was developed to specifically bind to and block the activity of the 165 amino acid isoform of vegf (vegf165), the major inducer of abnormal blood vessel growth and leakage in wet amd . Pegaptanib has a mean apparent half - life in the vitreous of 10 4 days (patel et al 2006), but to prolong activity at the site of action, the sugar backbone was modified to prevent degradation and the aptamer was covalently linked to two branched 20-kd polyethylene glycol moieties, which increases its half - life in the vitreous (ruckman et al 1998; drolet et al 2000). Vegf165 consists of a receptor - binding domain, which is found on all vegf isoforms, and a heparin - binding domain, which is unique to vegf165 (ferrara et al 2003). Pegaptanib binds to the heparin - binding domain, accounting for its specificity for vegf165, with an extremely high affinity (kd = 50 pm) (lee et al 2005) and inhibits the interaction of vegf165 with its type-1 and type-2 receptors . With cultured human umbilical vein endothelial cells, pegaptanib inhibited the binding, signal transduction, calcium mobilization, and cell proliferation mediated by vegf165 to an extent comparable with anti - vegf monoclonal antibodies (bell et al 1999). Based on its vast potential for the treatment of amd, diabetic retinopathy, and tumors, preclinical evaluation of pegaptanib was quickly undertaken to determine its safety and efficacy . Following intravitreal administration of pegaptanib into rhesus monkeys, there were no toxic effects, no change in intraocular pressure, and no immune response to the aptamer . There was a half - life of the aptamer in the vitreous of approximately 90100 hours, depending on the dose administered, and the compound remained fully active in the eye for at least 28 days following biweekly injections (drolet et al 2000). Subcutaneous and intravenous administration were also effective at maintaining adequate plasma levels (tucker et al 1999), providing a basis for subcutaneous delivery of the aptamer . In the miles assay, pegaptanib almost completely blocked vegf - mediated vascular leakage and inhibited corneal angiogenesis by 65% in a rat model and retinal neovascularization in a murine model of retinopathy of prematurity (eyetech study group 2002). In diabetic rats, pegaptanib significantly suppressed leukostasis and vascular leakage in both the early and late stages of the disease (ishida et al 2003). Trans - scleral delivery of pegaptanib was also achieved in rabbits by encapsulating the aptamer in poly(lactic - co - glycolic) acid (plga) microspheres (carrasquillo et al 2003). The drug was released over a period of 20 days and retained activity, providing a promising approach for the treatment of retinal and choroidal disorders with a dosing frequency of a minimum of every 6 weeks . The sustained release of pegaptanib was extended to several weeks using plga - based microspheres in rabbits (cook et al 2006). Abbreviations: nv, choroidal neovascularization; oir, oxygen - induced retinopathy; vegf, vascular endothelial growth factor . The initial clinical trials were previously reviewed (vinores 2003), but there have been several recent developments . Phase i trials with peg - conjugated pegaptanib sodium began in 1998 following us food and drug administration (fda) approval, making it the first aptamer to reach clinical testing . This study, conducted by eyetech pharmaceuticals, utilized dosages ranging from 0.25 to 30 mg / eye in 15 patients with wet amd and demonstrated stabilization or improvement of vision in 80% of patients at 3 months and 26.7% showed an improvement of 3 lines or more without any toxicity (eyetech study group 2002; guyer et al 2003). Eyetech followed with a phase ii study involving multiple intravitreal injections with or without photodymanic therapy (pdt), which was conducted with 21 patients with subfoveal cnv secondary to amd . In 87.5% of patients receiving only pegaptanib, vision stabilized or improved with 25% showing a 3 lines or greater improvement, whereas pdt alone was effective in only 50.5% with only 2.2% showing an improvement of 3 lines or more . The level of improvement reached 60% if pegaptanib and pdt were administered together (guyer et al 2003). Due to the potential for pegaptanib to provide a better alternative than laser photocoagulation for the treatment of exudative (wet) amd, the fda granted fast - track designation for phase iii clinical trials and these trials were underway by 2002 and involved 1186 patients at 117 centers (gragoudas et al 2004). Pegaptanib has also recently been approved for the treatment of wet amd in canada (trials launched in 2005) and it has been filed for approval in the european union, australia, switzerland, and brazil (eyetech pharmaceuticals, inc . Trials, doses of 0.3, 1.0, or 3.0 mg were administered and efficacy was demonstrated for all three doses without a dose - response relationship . Seventy percent of the patients who received 0.3 mg pegaptanib lost fewer than 15 letters of visual acuity compared with 55% in sham - injected controls (gragoudas et al 2004) and the improvement was maintained in a 1-year extension of the trials (siddiqui and keating 2005). Separate evaluations yielded similar results, with 78% of pegaptanib - treated patients showing loss of fewer than 15 letters of visual acuity compared with 54% in patients receiving usual care (vision clinical trial group 2005). The risk of severe loss of visual acuity (30 letters) was reduced from 22% in the sham - injected group to 10% in patients receiving 0.3 mg pegaptanib (gragoudas et al 2004). A separate evaluation found that patients receiving usual care were approximately 10 times more likely to suffer severe vision loss (29%) than those treated with pegaptanib (3%) (vision clinical trial group 2005). Significantly more patients maintained or gained visual acuity if they received pegaptanib (33% compared with 23% for sham - treated controls). Commencing 6 weeks after treatment, visual acuity was consistently better in pegaptanib - treated patients (gragoudas et al 2004). In a recent study involving 40 patients treated with 1 mg intravitreal pegaptanib every 6 weeks for a duration of 24 weeks, the thickness of the central retina decreased from 340 24 m to 280 20 m (p = 0.02) and vascular leakage, assessed by fluorescein angiograms, decreased from 100% to 54% while stable visual acuity was maintained (emerson et al 2006). Surprisingly, when a single eye was treated with pegaptanib, a significant macular thickness reduction was noted in the uninjected eye . This remote biological effect, possibly occurring through systemic absorption, raises concerns about pegaptanib s possible interference with physiological angiogenesis, such as coronary collateral vessel formation and wound healing (martin et al 2006). Abbreviations: pdt photodynamic therapy . In a previous evaluation of pegaptanib (vinores 2003), no treatment - related adverse effects were noted . Even at doses 3- to 10-fold higher than the recommended 0.3 mg / eye dose, pegaptanib had an excellent safety profile (patel et al 2006), but some adverse effects have recently been reported with the progression of the phase iii trials . Endopthalmitis occurred in 1.3% of patients, traumatic injury to the lens in 0.7%, and retinal detachment in 0.6%, accounting for the most serious adverse effects . Collectively, the adverse events accounted for severe loss of visual acuity in 0.1% of patients (gragoudas et al 2004). There was no evidence of a sustained increase in intraocular pressure following pegaptanib injection in either a short - term (hariprasad et al 2006) or a long - term setting (gragoudas et al 2004); however, three patients experienced severe eye pain that did not effect visual acuity within 2 hours of the injection (liggett et al 2006). Intraocular pressure was not elevated, only mild conjunctival inflammation around the injection site was noted, and the fundus examination was unchanged; therefore, the etiology of the pain was unclear . Also reported were two cases of severe systemic allergic responses in association with vitreous administration of pegaptanib (steffensmeier et al 2006). Other adverse effects reported following injection of pegaptanib include vitreous floaters, vitreous opacities, anterior chamber inflammation, reduced visual acuity, corneal edema, blurred vision, and dizziness (doggrell 2005; thomson centerwatch 2005). Pegaptanib treatment, alone, produces modest effects in the treatment of amd, but it may have added benefit when used in combination with other therapies . Intravitreal pegaptanib injections for amd results in stabilization of vision and a significant reduction in subretinal fluid thickness, but there were no significant anatomical changes in foveal or maximal retinal thickness, pigment epithelial detachment, total lesion thickness, cystoid macular edema, or cnv membrane thickness (ufret et al 2006), so combining pegaptanib with an alternative therapy may help to improve the outcome . In two animal models of ocular nv, pegaptanib in combination with pdt was more effective at inhibiting and promoting regression of nv than either was alone (ju et al 2006). Pegaptanib has also been used successfully in combination with pdt for the treatment of amd (guyer et al 2003; vann et al 2006). Anti - vegf therapies, such as pegaptanib, tend to be less effective at trying to promote regression of more established vessels than at treating amd in its early stages . In addition, pdt upregulates vegf, potentially leading to further complications . In both studies, the combination of pdt and pegaptanib treatment improved visual acuity in 60% of the patients, which exceeded the outcome of either mode of therapy alone, and there were no additional safety concerns with the combination therapy . Pegaptanib has also been used sucessfully with bevacizumab (avastin), which is a broader spectrum anti - vegf treatment, reacting with multiple isoforms rather than specifically with vegf165 . Preliminary results suggest that bevacizumab has the potential to improve the vision in patients who had previously been treated with pegaptanib and that pegaptanib can be used to maintain these gains while potentially minimizing the toxicity of a pan isoform vegf inhibitor, such as bevacizumab (tolentino et al 2006). Preliminary results in a separate study suggest that pegaptanib may also be useful when administered subsequent to bevacizumab (hughes and sang 2006). Patients with occult cnv associated with amd respond better to pegaptanib than do patients with classic cnv, raising speculation that another isoform of vegf may be responsible for the development of classic cnv (iyenger et al 2006) and possibly accounting for the added benefit of bevacizumab in conjunction with pegaptanib . Vegf has been identified as a key molecule in the development of ocular nv and vascular permeability, making it a good therapeutic target for the treatment of amd . Pegaptanib, an anti - vegf therapy, is the first agent to be used in clinical trials for amd and the first aptamer used in clinical trials . The encouraging preliminary results with animal and clinical trials prompted the fda to grant fast - track status for the treatment of amd . The phase iii clinical trials showed modest effects, primarily at stabilization of vision and reduction of subretinal fluid . Pegaptanib or other anti - vegf therapies were most effective against amd in the early stages and were not particularly effective against the more established vessels . When comparing different treatments for their efficacy in treating amd using the lineweaver - burke (lb) linear plot and correcting for differences in the initial visual acuity score (vas), pegaptanib, pdt, and anecortave acetate produced a similar slope and ranibizumab (lucentis), another anti - vegf therapy, appeared to be the most efficacious treatment, since it is the only treatment proven to reverse the slope of visual acuity on a lb plot . If all treatments are started at an initial vas of 60 letters, the expected final vas for ranibizumab would be 69.0 letters, for pdt 22.1 letters, for pegaptanib 18.4 letters, and for anecortave acetate 21.3 letters (shah et al 2006). Pegaptanib, however, shows greater efficacy when combined with another mode of therapy, such as pdt or bevacizumab, and these and other combination therapies may be the most promising therapeutic approaches for treating amd.
Cmbs are regarded as one of the indicators of cerebral small vessel disease, especially when located in the deep gray matter or infratentorial area . Risk factors associated with cmbs in the lobar area are different than those associated with deep cmbs . A different pathomechanism of cortical microangiopathy by amyloid deposition (i.e., cerebral amyloid angiopathy) is regarded as the major cause of lobar cmb . In addition, lobar cmbs are also predominantly observed in patients with a cardioembolic source of septic emboli, which may cause small mycotic aneurysms in their cerebral arteries . The prevalence of cmbs in stroke patients differs according to the subtype of ischemic stroke . The prevalence has been found to be highest in patients with small lacunar infarctions [9 - 11]; however, cmbs in other subtypes of stroke are also common . In fact, up to one - third of cardioembolic stroke (ce) cases demonstrate cmbs [14 - 16]. Considering that patients with ce require long - term anticoagulation, and that anticoagulation increases the risk of intracerebral hemorrhage, especially in patients with multiple cmbs, understanding the pathomechanism of cmbs according to ischemic stroke subtype or location may be important in determining the treatment strategy for secondary stroke prevention . In our current study, we investigated the risk factors and locations of cmbs in patients with different ischemic stroke subtypes, and the risk factors for cmbs in different locations . Ischemic stroke patients who were admitted within 1 week of stroke onset to the stroke center at asan medical center (seoul, korea) between january 2007 and january 2010 were retrospectively reviewed from a prospectively collected database . Among these cases, patients classified as having large artery atherosclerosis (laa), small vessel occlusion (svo), or ce were consecutively enrolled after excluding other etiologies . The ischemic stroke subtypes in this population were classified in accordance with the toast (trial of org 10172 in acute stroke treatment) system . Holter monitoring, and transthoracic and/or transesophageal echocardiography were performed in selected patients who (1) had a history of heart disease or arrhythmia on electrocardiography, (2) were young (<50 years of age), or (3) had any cortical lesion on diffusion - weighted image without evidence of the embolic source on magnetic resonance angiography . Patients classified as undetermined or other determined etiology and patients with poor quality image that were not amenable to analysis were excluded from the study . The requirement for obtaining informed consent was waived because of the retrospective nature of the analysis . The demographic characteristics of the study patients, including their vascular risk factors and previous use of antithrombotic agents (antiplatelet agent and anticoagulants) were collected from medical records . The risk factors included hypertension (defined as receiving medications for hypertension or blood pressure> 140/90 mmhg on repeated measurements), diabetes mellitus (defined as receiving medications for diabetes mellitus, fasting blood sugar 126 mg / dl or hba1c 6.5%, or a casual plasma glucose> 200 mg / dl), previous heart disease, hypercholesterolemia (defined as receiving cholesterol - reducing agents or an overnight fasting cholesterol level 240 mg / dl, 200 mg / dl triglycerides, or low density lipoprotein (ldl) cholesterol 160 mg / dl), and current cigarette smokin . Mri scans were obtained within one week of stroke onset using either a 1.5 t or 3.0 t mr imaging unit . All the enrolled patients underwent diffusion - weighted image, gre, and magnetic resonance angiography . The common parameters for diffusion - weighted image were a slice thickness of 5 mm, an inter - slice gap of 2 mm, 20 axial slices, and a field of view of 250 mm . The mri parameters for gre were a slice of thickness of 5 mm, an inter - slice gap of 2 mm, 20 axial slices, a field of view of 250 mm, a 400-ms repetition time, 30-ms echo time, 20 flip angle, and a 256192 matrix . Common magnetic resonance angiography parameters included a flip angle of 20, a 512512 matrix, and a field of view of 250 mm . Cmbs were defined as lesions with homogeneous round signal loss and a diameter 5 mm on gre . However, hypointense lesions within the subarachnoid space and areas of symmetrical hypointensity in the globus pallidus on gre were considered likely to represent adjacent pial blood vessels and calcifications, respectively . Cmbs located at the cortico - subcortical area of each lobe (i.e., frontal, parietal, temporal, or occipital) were regarded as lobar cmbs . Cmbs in the basal ganglia, thalamus, brainstem, and cerebellum were categorized as deep cmbs . Multiple cmbs that were distributed throughout both the lobar and deep areas were defined as diffuse cmbs . If at least one cmb existed at lobar area or deep area, the patient was regarded to have any lobar or any deep cmb . Classification of stroke subtype and detection of the cmbs were performed by two independent neurologists (y.y and h.s) who were blind to the clinical data, and any discrepancies were reevaluated at a consensus meeting . Pearson chi - square test, student t test, or mann - whitney test was appropriately used . In addition, multivariable logistic regression analysis was performed to estimate the independent contributions of various factors on the presence of cmbs in different stroke subtypes and locations . Variables were selected for entry into the model based on the results of univariable analysis (p0.2); svo group: age, hypertension and previous stroke history, laa group: hypertension and previous stroke history, ce group: hypertension and previous use of antithrombotic agents were entered . For locations, factors associated with any lobar, lobar, spss for windows was used for these analyses (version 18.0; spss inc ., ischemic stroke patients who were admitted within 1 week of stroke onset to the stroke center at asan medical center (seoul, korea) between january 2007 and january 2010 were retrospectively reviewed from a prospectively collected database . Among these cases, patients classified as having large artery atherosclerosis (laa), small vessel occlusion (svo), or ce were consecutively enrolled after excluding other etiologies . The ischemic stroke subtypes in this population were classified in accordance with the toast (trial of org 10172 in acute stroke treatment) system . Holter monitoring, and transthoracic and/or transesophageal echocardiography were performed in selected patients who (1) had a history of heart disease or arrhythmia on electrocardiography, (2) were young (<50 years of age), or (3) had any cortical lesion on diffusion - weighted image without evidence of the embolic source on magnetic resonance angiography . Patients classified as undetermined or other determined etiology and patients with poor quality image that were not amenable to analysis were excluded from the study . The requirement for obtaining informed consent was waived because of the retrospective nature of the analysis . The demographic characteristics of the study patients, including their vascular risk factors and previous use of antithrombotic agents (antiplatelet agent and anticoagulants) were collected from medical records . The risk factors included hypertension (defined as receiving medications for hypertension or blood pressure> 140/90 mmhg on repeated measurements), diabetes mellitus (defined as receiving medications for diabetes mellitus, fasting blood sugar 126 mg / dl or hba1c 6.5%, or a casual plasma glucose> 200 mg / dl), previous heart disease, hypercholesterolemia (defined as receiving cholesterol - reducing agents or an overnight fasting cholesterol level 240 mg / dl, 200 mg / dl triglycerides, or low density lipoprotein (ldl) cholesterol 160 mg / dl), and current cigarette smokin . Mri scans were obtained within one week of stroke onset using either a 1.5 t or 3.0 t mr imaging unit . All the enrolled patients underwent diffusion - weighted image, gre, and magnetic resonance angiography . The common parameters for diffusion - weighted image were a slice thickness of 5 mm, an inter - slice gap of 2 mm, 20 axial slices, and a field of view of 250 mm . The mri parameters for gre were a slice of thickness of 5 mm, an inter - slice gap of 2 mm, 20 axial slices, a field of view of 250 mm, a 400-ms repetition time, 30-ms echo time, 20 flip angle, and a 256192 matrix . Common magnetic resonance angiography parameters included a flip angle of 20, a 512512 matrix, and a field of view of 250 mm . Cmbs were defined as lesions with homogeneous round signal loss and a diameter 5 mm on gre . However, hypointense lesions within the subarachnoid space and areas of symmetrical hypointensity in the globus pallidus on gre were considered likely to represent adjacent pial blood vessels and calcifications, respectively . Cmbs located at the cortico - subcortical area of each lobe (i.e., frontal, parietal, temporal, or occipital) were regarded as lobar cmbs . Cmbs in the basal ganglia, thalamus, brainstem, and cerebellum were categorized as deep cmbs . Multiple cmbs that were distributed throughout both the lobar and deep areas were defined as diffuse cmbs . If at least one cmb existed at lobar area or deep area, the patient was regarded to have any lobar or any deep cmb . Classification of stroke subtype and detection of the cmbs were performed by two independent neurologists (y.y and h.s) who were blind to the clinical data, and any discrepancies were reevaluated at a consensus meeting . Pearson chi - square test, student t test, or mann - whitney test was appropriately used . In addition, multivariable logistic regression analysis was performed to estimate the independent contributions of various factors on the presence of cmbs in different stroke subtypes and locations . Variables were selected for entry into the model based on the results of univariable analysis (p0.2); svo group: age, hypertension and previous stroke history, laa group: hypertension and previous stroke history, ce group: hypertension and previous use of antithrombotic agents were entered . For locations, factors associated with any lobar, lobar, spss for windows was used for these analyses (version 18.0; spss inc ., during the study period, 1,357 patients within 7 days from ischemic stroke onset were registered to the database during the study period . Among them, 169 patients with undetermined etiology and 64 patients with other determined etiology were excluded . Of them, 1,124 patients were initially determined to be eligible to participate and were screened . However, 91 patients were excluded due to suboptimal image quality and a final study population of 1,033 patients was included . The ischemic stroke subtypes were laa (n=432; 41.8%), svo (n=304; 29.4%), and ce (n=297; 28.8%; figure 1). The demographic features and vascular risk factors were significantly different between patients with different ischemic stroke subtypes . Compared with laa or svo patients, most of the vascular risk factors, including hypertension, diabetes mellitus, hyperlipidemia, and smoking, were less frequent in ce patients . However, the proportion of ce patients who had previously used antithrombotic agents was significantly higher than the other subtypes (table 1). The prevalence and mean number of cmbs were significantly different between the various ischemic stroke subtypes: prevalence, svo (40.5%), ce (33.0%), and laa (24.8%; p<0.001; table 1); mean number of cmbs, svo (meanstandard deviation: 4.657.40), ce (3.093.49), and laa (3.123.63; p=0.041). As indicated in table 1 and figure 1, the location of the cmbs also differed according to the subtype of ischemic stroke (p=0.004). The presence of any lobar cmb was high in svo and ce patients, whereas deep cmb was frequently observed from svo patients (figure 2). Finally, the ratio of patients with any cmbs in the lobar area to that of deep area was relatively high in patients with ce (63/71; 88.7%) compared to svo (71/97; 73.2%) or laa (43/85; 50.6%) cases (figure 2). The concordance rate between the two investigators for the location of cmbs was 90.4%, and the kappa value was 0.71 . The clinical characteristics of patients with and without cmb for each ischemic stroke subtype are presented in table 2 . In patients categorized as svo or laa, the prevalence of hypertension and previous stroke history were higher in patients with cmbs than in those without . Patients with cmbs were older (66.011.0 vs. 63.113.0; p<0.05) than those without cmbs in the svo group . By multivariate analysis, hypertension (or=1.956 [95% confidence interval=1.0643.594]; p=0.031) and a previous history of stroke (or=1.929 [1.1253.309]; p=0.017) were independent risk factors for cmbs in svo patients . Similarly, hypertension (or=3.656 [1.7607.594]; p=0.001) and a previous history of stroke (or=1.663 [1.0362.669] p=0.035) were also independent risk factors for cmbs in laa patients . However, these factors were not associated with the presence of cmbs in ce patients . In terms of cmb location, the results of univariable analysis are demonstrated at table 3 . According to the results of multivariable analysis, the presence of strictly lobar cmb was independently associated with ce (or=1.85 [1.023.34]; p=0.042) and use of antithrombotic agents (or=1.80 [1.102.94]; p=0.019). Age (or=1.02 [1.001.04]; p=0.015) and hypertension (or=1.61 [1.082.40]; p=0.020) were independently associated with the presence of deep cmb . The presence of any lobar cmb was independently associated with the use of antithrombotic agents (or=1.97 [1.263.07]; p=0.003), whereas sex was the only factor associated with the presence of any deep cmb (male sex: or=0.50 [0.290.86]; p=0.013). During the study period, 1,357 patients within 7 days from ischemic stroke onset were registered to the database during the study period . Among them, 169 patients with undetermined etiology and 64 patients with other determined etiology were excluded . Of them, 1,124 patients were initially determined to be eligible to participate and were screened . However, 91 patients were excluded due to suboptimal image quality and a final study population of 1,033 patients was included . The ischemic stroke subtypes were laa (n=432; 41.8%), svo (n=304; 29.4%), and ce (n=297; 28.8%; figure 1). The demographic features and vascular risk factors were significantly different between patients with different ischemic stroke subtypes . Compared with laa or svo patients, most of the vascular risk factors, including hypertension, diabetes mellitus, hyperlipidemia, and smoking, were less frequent in ce patients . However, the proportion of ce patients who had previously used antithrombotic agents was significantly higher than the other subtypes (table 1). The prevalence and mean number of cmbs were significantly different between the various ischemic stroke subtypes: prevalence, svo (40.5%), ce (33.0%), and laa (24.8%; p<0.001; table 1); mean number of cmbs, svo (meanstandard deviation: 4.657.40), ce (3.093.49), and laa (3.123.63; p=0.041). As indicated in table 1 and figure 1, the location of the cmbs also differed according to the subtype of ischemic stroke (p=0.004). The presence of any lobar cmb was high in svo and ce patients, whereas deep cmb was frequently observed from svo patients (figure 2). Finally, the ratio of patients with any cmbs in the lobar area to that of deep area was relatively high in patients with ce (63/71; 88.7%) compared to svo (71/97; 73.2%) or laa (43/85; 50.6%) cases (figure 2). The concordance rate between the two investigators for the location of cmbs was 90.4%, and the kappa value was 0.71 . The clinical characteristics of patients with and without cmb for each ischemic stroke subtype are presented in table 2 . In patients categorized as svo or laa, the prevalence of hypertension and previous stroke history were higher in patients with cmbs than in those without . Patients with cmbs were older (66.011.0 vs. 63.113.0; p<0.05) than those without cmbs in the svo group . By multivariate analysis, hypertension (or=1.956 [95% confidence interval=1.0643.594]; p=0.031) and a previous history of stroke (or=1.929 [1.1253.309]; p=0.017) were independent risk factors for cmbs in svo patients . Similarly, hypertension (or=3.656 [1.7607.594]; p=0.001) and a previous history of stroke (or=1.663 [1.0362.669] p=0.035) were also independent risk factors for cmbs in laa patients . However, these factors were not associated with the presence of cmbs in ce patients . In terms of cmb location, the results of univariable analysis are demonstrated at table 3 . According to the results of multivariable analysis, the presence of strictly lobar cmb was independently associated with ce (or=1.85 [1.023.34]; p=0.042) and use of antithrombotic agents (or=1.80 [1.102.94]; p=0.019). Age (or=1.02 [1.001.04]; p=0.015) and hypertension (or=1.61 [1.082.40]; p=0.020) were independently associated with the presence of deep cmb . The presence of any lobar cmb was independently associated with the use of antithrombotic agents (or=1.97 [1.263.07]; p=0.003), whereas sex was the only factor associated with the presence of any deep cmb (male sex: or=0.50 [0.290.86]; p=0.013). Our present findings indicate that the prevalence, location, and risk factors associated with cmbs differ significantly according to ischemic stroke subtype . Cmbs were most frequently observed in svo patients, followed by ce and laa patients . The distribution of cmbs was mostly in deep areas in svo and laa patients, whereas the cmbs were found in the lobar area relatively more frequently in ce patients compared to svo and laa patients . The presence of cmbs was associated with hypertension and previous stroke history in svo or laa cases, whereas there was no significant association between those factors and the presence of cmbs in patients with ce . Prior use of antithrombotics was associated with the presence of any lobar cmb, which was relatively frequent in ce patients . The prevalence of cmb according to ischemic stroke subtype in our current series was consistent with the findings of previous reports . Not only the prevalence, but also the cmb location, differed according to ischemic stroke subtype . Svo patients more frequently demonstrated cmbs in deep areas, where the deep perforating arteries exist . Previously, cmbs located in deep areas were found to be associated with high blood pressure, renal impairment, and heavy alcohol consumption [21 - 23]. From our present study findings, cmbs in those areas are suggested to be caused by hypertensive arteriopathy, which affects the perforators . Cmbs in lobar areas have been associated with smoking, apoe 4, and low cholesterol levels . Recently, several studies revealed lobar cmbs in specific lobar areas to be associated with various degenerative diseases (e.g. Alzheimer s disease, parkinson s disease and diffuse lewy body disease) [26 - 28]. However, age, which is the strongest risk factor for degenerative diseases, was not found to be associated with lobar cmbs in our present study with stroke population . Instead, we found that ce and the previous use of antithrombotics were associated with the presence of lobar cmb . Cmbs have previously been associated with the use of anticoagulants, and this relationship was reported to be stronger in patients from the general population with a greater fluctuation of prothrombin time . Furthermore, among patients with ischemic stroke, cmbs were found to be common in patients with atrial fibrillation, and the use of antithrombotics was independently associated with the presence of lobar but not deep cmbs . In our present study, lobar cmbs were more frequent in ce patients, and the presence of lobar cmb was associated with a prior use of antithrombotics . Patients with ce generally show small asymptomatic cortical infarctions, higher spontaneous recanalization rates, and concomitant hemorrhagic transformations . Considering that patients under oral anticoagulation are more likely to develop cmbs, the use of antithrombotics may enhance micro - hemorrhages occurring as a result of recanalization of small arterioles obstructed by an asymptomatic embolus originating from the heart [32 - 34]. The characteristics of lobar cmbs developed through such a mechanism may differ from cmbs caused by degenerative disease . First, due to its retrospective design, there was a risk of selection bias, which could have impacted on our findings of cause and effect . However, our patients were consecutively recruited from a single center, and most of these cases underwent brain mri using an established protocol . Second, we used both 1.5 t and 3.0 t mri, although 1.5 t mri is inferior for the detection of cmbs . Notwithstanding the above limitations, our present study demonstrated that the distribution and risk factors associated with cmbs differed by stroke subtype, especially in patients with ce compared with svo or laa . Our results suggest different pathogenic mechanisms for the development of cmbs of each subtype and that the previous use of antithrombotic agents is related to cmb location.
Dna alkylating agents have been used in the clinic for more than 60 years for the treatment of cancer and are still considered first - line medication . Their mode of action involves covalent modification to dna by forming genotoxic dna adducts that can interfere with replication of cells, especially in rapidly dividing cancer cells . However, a drawback is the typically poor selectivity of these drugs to target cancer cells, which usually results in a wide range of side effects in normal cells of treated patients and arises from processes that are often poorly understood . The use of drug combinations is a common strategy to limit the dose required for each single agent . In addition, reduction of unwanted toxicity can be achieved by developing drugs that are selectively activated by conditions prevalent in cancer, but not in normal tissues such as hypoxia, aberrant expression of metabolic enzymes, or oxidative stress . Pr104a is an experimental dna - alkylating hypoxia - activated prodrug (hap). This drug is administered as the corresponding phosphate ester pr104, which is systemically hydrolyzed to the alcohol prodrug pr104a . Pr104a undergoes metabolic nitroreduction to generate cytotoxic hydroxylamine (pr104h) and amine (pr104 m) metabolites (scheme 1). One - electron reductases such as cypor, mtrr, ndor1, and nos2a mediate this activation in a process that is inhibited by the presence of oxygen . In fact, the initial radical that results from this reduction (1, scheme 1) can be reoxidized by oxygen, whereas under hypoxic conditions, further reduction generates the two major metabolites pr104h and pr104 m . These metabolites induce cross - links in dna . It has been found recently that pr104a also can be activated in an oxygen - independent manner by the two - electron aldo - keto reductase 1c3 (akr1c3, scheme 1). Evidence for pr104a activation by akr1c3 includes correlation of protein expression and oxic metabolism of pr104a and enhancement of pr104 antitumor activity in akr1c3-negative tumor xenograft models engineered to overexpress akr1c3 . In a recent study, preconditioning of ht-29 colon cancer cells with a low nontoxic dose of sulforaphane (sf), an isothiocyanate from cruciferous vegetables with the ability to induce xenobiotic - metabolizing and antioxidant enzymes, up - regulated the level of 18 proteins . Among them, akr1c3 was seven - fold more abundant in sf - treated cells . Sf - preconditioning prior to pr104a treatment sensitized ht-29 cells to pr104a and decreased the ec50 for cell death by a factor of 3.6 but did not sensitize the immortalized, nontransformed normal human colonic epithelial cells (hcec). The study focused on the relationship between akr1c3 levels and cytotoxicity, but the impact of sf on dna damage induced by pr104a was not investigated . There is clear evidence that dna interstrand cross - links contribute to pr104a cytotoxicity, as demonstrated by increased sensitivity of cell lines with genetic defects in cross - link repair and correlations between cytotoxicity and cross - link formation as determined by the alkaline comet assay in cell lines in vitro and in xenografts . However, these studies also demonstrated that in cell lines with low rates of metabolic activation of pr104a, cytotoxicity is not accounted for by cross - link formation . Thus, more direct methods for quantitation of dna damage by pr104a are needed to better understand its mode of toxicity . As an initial step, we recently developed a high - resolution / accurate - mass (hram) lc ms dna adductomic method for screening for pr104a - derived dna adducts . Both mono and cross - linked dna adducts induced by the two metabolites, pr104h and pr104 m, and by direct alkylation of dna by pr104a were identified in dna extracted from cancer cells treated with pr104a . However, to our knowledge, an approach for quantifying pr104a - derived dna adducts in biological samples does not exist . Studies involving the application of a strategy for quantifying pr104a adducts are expected to lead to improved understanding of the selectivity factors driving the efficacy of metabolism - activated dna alkylating agents . Therefore, a goal of this study was to quantify pr104a - derived dna adducts in cells and evaluate how factors that modulate the responsiveness of cells are reflected in dna adduct profiles . The strategy was based on the creation of a stable isotope - labeled adduct mixture (silam) as a reference standard by enzymatic reduction of pr104a in the presence of dna . It was used as an internal standard for relative quantitation of adducts by stable isotope dilution mass spectrometry . The silam was generated and characterized, and its use as reference standard was validated by measuring adduct levels in purified dna and in dna extracted from cells treated with pr104a . Lc ms analysis using the silam was carried out via a selected reaction monitoring (srm) approach targeting 19 dna adducts induced by pr104a and its metabolites pr104h and pr104 m previously detected in cells via dna adductomic screening . We used the silam - srm approach to test the hypothesis that enhanced cytotoxicity observed by sf - preconditioning of ht-29 cells is induced by increased level of dna adducts by measuring their levels in colon cancer cells preconditioned with sf . Calf thymus dna (ctdna) was purchased from worthington biochemical corporation (lakewood, nj). Akr1c3 was purchased from united states biological life sciences (salem, ma). The reference standard [pyridine - d4]o-[4-(3-pyridyl)-4-oxobutyl-1-yl]-2-deoxyguanosine (d4-o - pob - dg) was prepared as previously described (isotopic purity, 98 at . Cell culture medium and supplements were purchased from invitrogen (life technologies, switzerland). All solvents used for high performance liquid chromatography (hplc) and ms analysis were of the purest grade commercially available (99.9%). Ht-29 cells were obtained from the leibniz - institute dsmz gmbh in january 2012 (braunschweig, germany). Ht-29 cells were grown in dmem medium with glutamax containing 10% fetal bovine serum and 1% penicillin / streptomycin . Immortalized normal diploid human colonic epithelial cells (clone hcec1ct, abbreviated hcec) were obtained in august 2011 from prof . Jerry shay and grown in a humidified incubator containing 5% co2 at 37 c . The two cell lines have regularly been confirmed to be mycoplasma free by a luminescence detection assay (mycoalert mycoplasma detection kit, lonza, switzerland). Full details regarding cytotoxicity analysis, reactions of ctdna with pr104a or d4-pr104a, validation experiment, and quantitation of dguo by hplc for this study can be found in the supporting information . Cells were seeded in six - well plates at a density of 5.0 10 cells / well and were allowed to attach overnight . Afterward, the medium was replaced with fresh medium containing a final concentration of 2.5 m sf or 0.1% dmso (solvent control). After 48 h, the medium was removed, the wells were washed with pbs, and cells were treated with medium containing a final concentration of 150 m pr104a or 0.1% dmso (solvent control) and incubated at 37 c for 4 h. thereafter, the dna from intact cells was isolated by extraction with a dna isolation kit for cells and tissues (roche, switzerland). In short, cells were washed with pbs and scraped to one side of the well using a scraper . Lysis buffer (750 l) was added to each well, cell lysates were collected and sonicated with a vibra - cell sonicator (sonics, ct) using the following conditions: 40% amplitude, 15 cycles at 10 s on and 15 s off . One microliter of proteinase k solution was added to the cell lysates, and samples were incubated for 1 h at 65 c . Samples were allowed to cool to room temperature, 50 l of rnase solution was added, and samples were incubated at 37 c for 1 h. care should be taken in removing rna from the sample since adducts from the bases common to dna and rna are indistinguishable . A sample of 330 l of protein precipitation solution was added and samples mixed vigorously, cooled on ice for 5 min, and centrifuged at 16 900 g for 4 min to form a protein pellet . Supernatants were transferred in clean 2 ml eppendorf tubes, and 1 ml cold isopropanol was added . Samples were gently mixed until the dna pellet was visible and centrifuged at 4 c, 16 900 g for 10 min . Supernatants were removed without disturbing dna pellets, which were washed with 1 ml cold etoh by centrifugation . The resulting dna pellets were allowed to dry at room temperature for 30 min . Dna concentrations of the resulting solutions were determined with a nanodrop 1000 spectrometer (thermo scientific, waltham, ma). The 10 mm tris - hcl/5 mm mgcl2 buffer (ph 7) was added to a final volume of 900 l . Samples were spiked with the silam (1.5 l) and thermally hydrolyzed at 80 c for 1 h. the sample containing the highest amount of dna was used as reference to calculate the units (u, corresponding to the amount of the enzyme that catalyzes the conversion of 1 mol of substrate per min) of enzymes for enzyme hydrolysis . Then 600 u / mg dna of dnase i was added, and samples were incubated overnight at 37 c . Afterward, additional dnase i (600 u / mg dna) was added together with phosphodiesterase i (20 mu / mg dna) and alkaline phosphatase (240 u / mg dna), followed by incubation at 37 c overnight . The enzymes were removed by centrifugation using a centrifree ultrafiltration device (mw cutoff 30 000 atomic mass unit (amu); merck millipore, cork, ireland). An aliquot (60 l) was taken from each sample for the analysis of deoxyguanosine (dguo) by hplc . A solution of the same buffer and enzymes was used as a negative control . Hydrolysates were purified by solid - phase extraction (spe) on a strata - x cartridge (30 m, phenomenex, torrance, ca). The samples were loaded on the cartridge, washed with 1 ml of 40% ch3oh, and eluted with 600 l of 80% ch3oh in h2o . Prior to lc ms analysis, samples were dissolved in 20% ch3oh in h2o to a final volume of 10 l . Corresponding to dna amounts ranging from 18 g (cell samples) or 1550 g (ctdna samples), 0.5, 2, or 5 l of hydrolyzed, spe - purified, and reconstituted sample was injected onto a nanoacquity uplc (waters co., milford, ma) system equipped with a 5 l injection loop . Separation was performed with a capillary column (75 m i d, 10 cm length, 15 m orifice) created by hand packing a commercially available fused - silica emitter (new objective, woburn ma) with 5 m luna c18 bonded separation media (phenomenex, torrance, ca). The flow rate was 1000 nl / min for 5.5 min, then decreased to 300 nl / min with a 50 min linear gradient from 250% ch3cn in 5 mm nh4oac aqueous buffer (ph 5.5), an increase to 98% ch3cn in 3 min, with a 2 min hold and a 5 min re - equilibration at 1000 nl / min at 2% ch3cn . The injection valve was switched at 5.5 min to remove the sample loop from the flow path during the gradient . Data - dependent cnl - ms scanning with nanoelectrospray was carried out using an ltq orbitrap velos instrument (thermo scientific, waltham, ma). The nanoelectrospray source voltage was 2.0 kv, and the capillary temperature was 350 c . The ion focusing and transfer elements of the instrument were adjusted for maximum signal intensity by using the automated instrument tuning feature while the background ion signal of m / z 371.1 (decamethylcyclopentasiloxane) was monitored to create the tune file used for data analysis . Data - dependent ms analysis was performed with repeated full scan detection followed by ms acquisition and constant neutral loss triggering of ms fragmentation . Full scan (2002000 da) detection was performed using the orbitrap detector at a resolution of 60 000 (at m / z 400) with one microscan (one mass analysis followed by ion detection), automatic gain control (agc) target settings of 1 10, and a maximum ion injection time setting of 100 ms . Ms fragmentation was performed in the ion trap on the three most intense full scan ions listed in a parent mass list with orbitrap detection at a resolution of 7500, agc of 2 10, one microscan, maximum ion injection time of 100 ms, and full scan injection waveforms enabled . The parent mass list was composed of 298 masses corresponding to [m + h] ions of anticipated mono and cross - linked dna adducts from reaction of the four dna bases with pr104a, pr104h, pr104 m, or the corresponding d4-analogues . Ms fragmentation parameters were as follows: 3 amu isolation width, normalized collision energy of 35, activation q of 0.25, and activation time of 10 ms . Data - dependent parameters were as follows: triggering threshold of 10 000, repeat count of one, exclusion list size of 500, exclusion duration of 60 s, and exclusion mass width of 5 ppm . Ms hcd fragmentation (2 amu isolation width, normalized collision energy of 35, activation time of 0.1 ms) with orbitrap detection at a resolution of 7500 was triggered upon observation of neutral losses (5 ppm) of 116.0474, 151.0494, 135.0545, 126.0429, and 111.0433 amu between the parent ion and one of the 50 most intense product ions from the ms spectrum, provided a minimum signal of 1000 was observed . The following ms parameters were used: one microscan, agc target setting 2 10, maximum ion injection time of 100 ms . All spectra were acquired using the background ion signal of m / z 371.10124 amu (decamethylcyclopentasiloxane) as a lock mass . Instrument sensitivity was checked before each analysis by injection of 10 fmol of labeled standard (d4-o - pob - dg) and by integration of the observed peak in the extracted ion chromatogram of the exact mass of its parent ion . For srm scanning, samples were analyzed by nanoelectrospray in srm mode on a tsq vantage instrument (thermo scientific, waltham, ma). The nanoelectrospray source voltage was 1.6 kv, and the capillary temperature was 300 c . Q2 cid gas pressure, 1.0 mtorr; collision gas, argon; scan width, m / z 0.100; scan time, 0.050 s; collision energy, 20 v (base adducts) or 15 v (nucleoside adducts); q1 peak width, 0.70 amu; and q3 peak width, 0.70 amu . Mass transitions monitored were m / z 446.2311.1, m / z 462.2311.1, m / z 476.2341.1, m / z 492.2341.1, m / z 494.1359.1, m / z 510.1359.1, m / z 554.1403.0, m / z 562.2446.2, m / z 563.2428.1, m / z 579.2428.1, and 579.2444.1, m / z 592.2476.1, m / z 595.2444.1, m / z 608.2492.2, m / z 611.3460.1, m / z 625.2474.2, m / z 695.3579.2, m / z 711.3595.2, and m / z 741.3625.2, and the 19 isotope labeled corresponding standards (+ 4 amu). The tune file used for the analysis was created by maximizing signal at conditions similar to those present during dna adduct elution (300 nl / min, 50% ch3cn, mass range 400800 amu). Instrument sensitivity was checked before each analysis by injection of the same sample containing seven pr104a - induced dna adducts and by comparison of the peak areas . A t test was used to verify whether sf - preconditioned and control samples were statistically significantly different from each other (graphpad prism 6). The silam to be used for relative quantitation was generated by reacting purified dna from calf thymus (ctdna) with d4-labeled pr104a in the presence of akr1c3 and nadph . Following a 24 h reaction, the dna was precipitated, the residual d4-pr104a removed by chloroform / isoamyl alcohol extraction, the dna hydrolyzed, and the hydrolysate enriched by solid phase extraction . The resulting mixture then was characterized by a previously reported hram lc ms dna adductomic approach . Briefly, repeated full scan detection followed by ms acquisition and constant neutral loss triggering of ms fragmentation (data - dependent scanning) to confirm the presence of a dna adduct was performed by nanoelectrospray on an ltq orbitrap velos . Data - dependent ms fragmentation was performed on the three most intense full scan ions listed in a parent mass list composed of d4-labeled masses of the [m + h] ions of anticipated mono and cross - linked dna adducts induced in the four dna bases by pr104a, pr104h, and pr104 m . Ms fragmentation was performed upon observation of the accurate mass neutral loss of the four dna bases (g, 151.0494 amu; a, 135.0545 amu; t, 126.0429 amu; and c, 111.0433 amu) or of the 2-deoxyribose (116.0474 amu). A total of 33 isotope - labeled mono and cross - linked dna adducts induced by pr104a, pr104h, and pr104 m were detected in the silam by the presence of an ms fragmentation event (figure 1, base peak accurate mass extracted ion chromatograms for the 33 detected adducts are shown in figure s1 in the supporting information). Their abundances in the silam, estimated by integration of the peak areas, varied by three orders of magnitude between the most to the least abundant adducts (figure 1). For example, the deaminated nucleoside monoadduct that resulted from cytosine alkylation by pr104a (m / z 635.1245, analyte 29 in figure 1) was among the less abundant adducts, whereas the hydrolyzed monoadduct that resulted from n7-guanine alkylation by pr104a (m / z 496.1837, analyte 7 in figure 1) was among the most abundant adducts present . D4-labeled pr104a dna adducts detected by ms fragmentation present in the stable isotope - labeled adduct mixture (silam). The silam - based method for relative quantitation of pr104a - derived dna adducts was validated by comparing calculated and measured dna adduct concentrations in samples obtained by diluting a single ctdna sample that had been reacted with pr104a into four samples containing increasing amounts of dna . All samples were spiked with the same amount of the silam, processed as described in the materials and methods section, and analyzed by lc results of this quantitation validation experiment involving purified dna are shown in figure 2 for a representative dna cross - link adduct (m / z 625) in samples analyzed with two different dna adductomic approaches: the hram lc ms approach and a new developed srm approach targeting 19 dna adducts induced by pr104a, pr104h, or pr104 m . For the pr104a - induced cross - linked dna adduct, the slope and r estimates were extrapolated and a very good correlation between calculated dna concentration and measured adduct concentration was found for both lc ms approaches used (figure 2). In addition to the cross - link adduct of m / z 625, 8 versus 12 additional adducts were present in the ctdna samples and quantified by the hram and srm approaches, respectively (structures, correlation plots, slopes, and r values for these adducts can be found in figures s2s4 in the supporting information). These adducts comprised mono and cross - linked adducts induced by the two metabolites or by direct alkylation by pr104a . For all these adducts and for both the analytical approaches (hram vs srm), as expected, a very good correlation was found between calculated dna and measured adduct concentrations (slope and r estimates ranged from 0.921.12 and 0.971.00 for the hram validation, and from 0.981.16 and 0.891.00 for the srm targeted validation, figures s2s4). Validation of the silam for relative quantitation of the pr104a - derived dna adducts (shown here for the dna cross - link with m / z 625 as a representative example) in purified dna treated with pr104a (100 m final concentration) for 24 h. analysis was performed with both a high - resolution analysis (hram) and with a nominal mass targeted analysis (srm). Because of the higher number of adducts detected and the ease of use and sensitivity of quantitation of triple quadrupole instrumentation operating in srm mode, we decided to use this system with the silam for relative quantitation . Thus, the accuracy of quantitation of this approach was evaluated for dna extracted from pr104a - treated cells . In an analogous experiment to that performed with purified dna, ht-29 cells were treated with pr104a (500 m final concentration), and the dna was extracted and divided into two samples with one containing 50% more dna than the other . These samples were spiked with the same amount of the silam, the dna adducts enriched by solid phase extraction, and samples were analyzed by nanolc a total of eight dna adducts induced by pr104a and pr104 m were quantified . Deviations of the measured dna adduct concentrations from the calculated dna amounts were between 0.6 and 5.5% (table s1). The newly developed silam approach was used to compare levels of pr104a - derived dna adducts in colon cancer cells preconditioned with sf . First, the experiments demonstrating increased sensitivity of ht-29 cells for pr104a treatment upon sf - preconditioning and the lack of alteration in sensitivity of hcec cells, which was observed previously, were repeated, and the results confirmed (figure s5). There was an increase in sensitivity of ht-29 cells toward pr104a (ec50 value 20.5 m vs 7.9 m for the nontreated vs sf - treated cells), which represented a 2.6-fold decrease, while there was no change in the case of hcec cells (figure s5). However, the relationship between adduct formation and the positive interaction of sf and pr104a was unknown . Pr104a - derived dna adducts were therefore quantified in ht-29 and hcec cells preconditioned with a sf concentration corresponding to the ec10 value derived for sf in ht-29 cells when individually tested . The analysis was performed by spiking the samples containing dna extracted from ht-29 or hcec cells preconditioned with sf and treated with pr104a with the silam prior to hydrolysis and dna adduct enrichment by solid phase extraction . The analysis was performed in srm mode targeting 19 pr104a-, pr104h-, and pr104m - induced dna adduct masses . The relative amount of each dna adduct was calculated by dividing the ratio of the peak areas corresponding to the unlabeled and labeled adducts by the total dna concentration of each sample . The total dna was determined by quantitation of 2-deoxyguanosine (dguo) by hplc . Negative control samples were samples containing buffer and enzymes used for the dna hydrolysis and a sample containing dna extracted from cells treated with sf only . None of the 19 targeted adducts was observed in these samples . A total of seven mono and cross - linked dna adducts resulting from pr104a, pr104h, or pr104 m were observed and quantified in both cell lines (figure 3). Some adducts were detected in both cell lines (m / z 462, 711, 492, 510, 625, and 741), whereas two adducts could be detected in one cell line only (m / z 695 in ht-29, and m / z 476 in hcec, figure 3). Dna adducts detected included both mono and cross - linked adducts from pr104a (m / z 476, 492, 510, 625, and 741) and its reactive pr104h and pr104 m metabolites (m / z 462, 695, and 711, figure 3c). Sf - preconditioning of ht-29 cells increased the amount of dna adducts induced by the two metabolites pr104h and pr104 m, but not of the dna adducts resulting from direct alkylation by pr104a (figure 3a). This increase was statistically significant for two of the metabolite - induced dna adducts quantified in ht-29 cells (figure 3a). On the other hand, no alteration of metabolite - induced dna adduct levels upon sf - preconditioning was observed in hcec cells (figure 3b). These data support a direct role of dna adducts in pr104a toxicity and, moreover, support a mechanistic model for the basis of how sf - preconditioning sensitizes ht-29 cells . Pr104a - derived dna adduct levels and fold change relative to sf - untreated controls in (a) ht-29 and (b) hcec cells upon sf - preconditioning versus controls . Asterisks above the bars represent statistical significance (t test, p <0.05), whereas nd represents nondetects . (c) proposed structures of pr104a - derived dna adducts quantified in ht-29 and hcec cells . A potential strategy to further our understanding of the mechanism of action of the prodrug pr104 and to support biomarker - based personalized strategies for its clinical use involves using dna adducts as drug - specific biomarkers of efficacy . Dna adducts have been used as mechanism - based biomarkers of exposure to carcinogens for hazard identification and risk assessment and for the estimation of human exposure to occupational, environmental, and dietary chemicals . Examples of anticancer drug classes for which the relationship between dna adducts and response were studied in vitro and in vivo include platinum - based drugs, nitrogen mustards, and minor groove binding agents . The advantage of monitoring drug dna adducts as biomarkers of efficacy relies on their drug - specificity and stability in dna . The challenge relies on the ability to identifying and quantifying them in biological samples since dna adducts are usually present in animal and human tissues in extremely low abundance compared to unmodified nucleobases (on the order of 0.0110 adducts per 10 unmodified nucleobases if induced by carcinogens, and on the order of 11000 adducts per 10 unmodified nucleobases if induced by anticancer drugs). In this study, a dna adductomic approach for the quantitation of previously detected pr104a - derived dna adducts in cells was developed and validated for allowing relative quantitation of pr104a - dna adducts in purified dna and in dna extracted from cells treated with pr104a . With the developed approach, dna adduct levels were measured in cells preconditioned with the bioactive food compound sulforaphane (sf), and the relationship between pr104a - derived dna adducts and cell viability response in vitro was investigated . We found a 2.4-fold increase in the level of dna adducts induced by the two metabolites pr104h and pr104 m in sf - preconditioned ht-29 cells . This increase was in good agreement with the 2.6-fold increase in cytotoxicity in ht-29 . To circumvent limitations to rapid development and validation of dna adduct monitoring strategies for clinical applications, we used an unconventional strategy for adduct quantitation that does not require synthesis of labeled standard for each adduct, but rather involves the creation of a stable isotope - labeled adduct mixture (silam). Quantitation of dna adducts usually involves spiking the samples at an early stage of the sample preparation with a chemically synthesized stable isotope - labeled internal standard of a dna adduct of interest . The isotope - labeled internal standard accounts for losses occurring during sample preparation, ion suppression from the sample matrix, and instrument variability during lc ms analysis . Addition of a known concentration of the stable isotope labeled standard allows one to calculate the accurate concentration of the adduct of interest in the sample, which results in absolute quantitation . Absolute quantitation approaches depend on the availability of stable isotope labeled internal standards for adducts of interest, the synthesis of which can be time - consuming and challenging depending, for example, on adduct structure and stability and on the cost of the stable isotope - labeled starting material . The results described herein demonstrate that using a silam reference standard can allow for the simultaneous relative quantitation of several pr104a - derived dna adducts in the same sample and account for adduct losses during sample preparation and ion suppression from sample matrix during lc ms analysis . On the basis of triggered ms fragmentation events for neutral loss of a dna adduct feature, and absence of this fragmentation event in the control samples, the silam contained 33 detectable isotopically labeled mono and cross - linked dna adducts induced by pr104a including adducts ascribed to alkylation by the metabolites pr104h and pr104 m (figure 1). The assignment of adducts as pr104a versus pr104h or pr104 m adducts was on the basis of their accurate masses and fragmentation patterns . The composition of the silam suggests the potential for deriving relative amounts of up to 33 pr104a - derived dna adducts simultaneously depending on whether they are present in biological samples . The use of silam for relative quantitation of pr104a - derived dna adducts was demonstrated by the strong correlation between calculated and measured dna adduct concentrations in a validation experiment involving pr104a - treated purified dna and dna extracted from pr104a - treated cells (figures 2 and s2s4, and table s1). The hram orbitrap analysis used for the initial pr104a - adduct discovery work also provided good relative quantitation in purified dna samples spiked with silam, in which nine adducts from the list of target adducts were quantified . This result is comparable to the 13 adducts measured by triple quadrupole srm - targeted analysis and is encouraging for future combined adduct discovery / relative quantitation investigations of this type . However, the silam triple quadrupole srm - targeted approach provided for relative quantitation of more of the targeted adducts and is a simpler, more widely available technology and therefore was chosen for the evaluation of pr104a - derived adducts in treated cells . Its accuracy and precision was evaluated for dna extracted from pr104a - treated cells, and the results further supported the suitability of the silam for relative quantitation of pr104a - derived dna adducts (table s1). The silam - srm approach has the potential to be used for monitoring dna adducts induced by dna alkylating drugs in patient samples . For this purpose, biological samples such as tumor biopsy, circulating cancer cells, and surrogate tissues could be collected from patients undergoing chemotherapy and dna adducts analyzed by silam - srm . Alternatively, the dna adducts can be quantified by the silam - srm approach in peripheral blood mononuclear cells or tumor biopsy isolated from patients treated with a microdose of the dna alkylating drug, whereas another potential scenario for sensitivity testing might involve ex vivo exposure of cancerous or normal surrogate cells to the dna alkylating drug with evaluation of adduct formation . We further evaluated the method in the context of characterizing dna adduct profiles in colon cancer cells preconditioned with sf prior to pr104a treatment . We previously reported that the increase in pr104a activity in ht-29 cells upon preconditioning with sf results from an increase in abundance and activity of the enzyme akr1c3 . Increase in gene expression of akr1c3, as for the other xenobiotic - metabolizing and antioxidant enzymes modulated by sf, is mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2). Thus, sf reacts with cysteine residues of the nrf2 repressor keap1, which results in nuclear translocation of nrf2 and binding of the transcription factor to dna . Higher amount of akr1c3 is thought to increase the level of drug activation and the production of reactive hydroxylamine (pr104h) and amine (pr104 m) metabolites, as demonstrated previously by analysis of the metabolites by lc the expectation was that sf - induced akr1c3 would lead to increased metabolite - induced dna adducts; on the other hand, dna adducts resulting from direct alkylation by pr104a should not depend on the level of akr1c3 . Therefore, we expected the levels of direct adducts to be invariant between sf - preconditioned and directly treated cells . This model was indeed supported by the results of this study, in which preconditioning ht-29 cells with sf led to an increase in the levels of dna adducts induced by the two metabolites pr104h and pr104 m but did not affect the level of dna adducts resulting from direct alkylation by pr104a (figure 3a). The average increase in metabolite - induced dna adducts relative to sf - untreated control in ht-29 cells (2.4 fold) matched closely the 2.6-fold decrease observed in the ec50 value derived from the cell viability dose response curve in ht-29 cells (figure s5). Among the three sf - modulated metabolite - induced dna adducts quantified in ht-29 cells, the one with m / z 462 was attributed to a hydrolyzed monoadduct induced by the reaction of pr104 m with a guanine, whereas the other two adducts with m / z 695 and 711 were attributed to cross - linked adducts induced by pr104h with two adenines, and by pr104 m with two guanines, respectively (figure 3c). An evaluation of the individual contribution of these adducts to cytotoxicity and therefore a conclusion regarding which of these adducts are more biologically relevant for the observed cytotoxicity is challenging, due to the heterogeneity of dna alkylation, that is, it is not possible to induce and observe the cytotoxic effects of one adduct at a time, rather all are formed in concert . Despite the ability of interstrand cross - links to prevent dna strand separation and block dna replication and transcription, their formation is much lower than that of monoadducts . Therefore, the overall cytotoxicity may result from a combination of effects from mono and cross - linked adducts, and further research outside the scope of the current study, such as using cells with the selective capacity to remove particular adducts, would be of interest to resolve this open question . Finally, it was anticipated that there would be no change in adduct levels when hcec cells were preconditioned with sf given the slight akr1c3 induction and no significant increase in akr1c3 activity in these nontumorigenic colonic mucosal cells . Again, this assertion could be confirmed (figure 3b). To investigate reasons for different cytotoxicity responses in ht-29 versus hcec cells, erzinger et al . Measured the cellular uptake of sf but found no significant differences in sf uptake between the two cell lines . Therefore, one may speculate that differences in induction of akr1c3 in ht-29 and hcec cells result from differences in the level of nrf2, or in the ability to translocate nrf2 into the nucleus to reach dna . Nevertheless, the absence of change in adduct levels in hcec cells upon sf - preconditioning measured in this study further supports the causative relationship between induction of pr104a - derived adducts and cytotoxicity . In conclusion, we established in this study evidence for a direct relationship between sf - preconditioning of cells, the abundance of dna adducts derived from the experimental nitrogen mustard prodrug pr104a, and pr104a cytotoxicity in a cancer and in a noncancerous cell line . This insight was enabled by the development of a stable isotope - labeled dna adductomic approach involving srm data acquisition for relative quantitation of dna adducts induced by pr104a in cells . The analytical approach based on nanolc ms - srm targeting and use of a silam reference mixture was useful to detect changes in the abundance of pr104a - derived dna adducts at pharmacologically relevant levels . The results of the quantitation of pr104a - derived dna adducts in cell lines suggest that these adducts contribute to pr104a potency, underlying the possibility of using them as biomarkers of efficacy to improve its chemotherapeutic selectivity and to stratify patients on the basis of their susceptibility to the drug . Moreover, the general dna adductomic - silam approach could be adapted for relative quantitation of dna adducts induced by any dna alkylating agent using a stable isotope - labeled analog of the agent, but without preparation of individual labeled adducts, as a tool to support drug discovery and improve the efficacy of existing anticancer drugs.
Appendix tumours are relatively rare cancers . In the examination of specimens after appendectomy, appendix cancer is observed with 1% incidence . According to the national cancer institute, using the surveillance, epidemiology, and end results (seer) data, appendix cancer accounts for 0.4% of all gastrointestinal tumours . Well - differentiated neuroendocrine tumours, traditionally referred to as carcinoid tumours, are the most frequently observed kind . Other cancers of the appendix are mucinous cystic adenocarcinoma, adenocarcinoma, goblet cell carcinoma, lymphoma, and paraganglioma . Generally, in the histopathological examination that is done after appendectomy performed due to acute appendicitis, carcinoid tumours are detected incidentally with a frequency of 0.30.9% [3, 4]. In many studies, in appendiceal neuroendocrine neoplasms (nen) smaller than 1 cm, with invasion up to the subserosa or mesoappendiceal invasion up to 3 mm, and clear surgical margins, no further risk of recurrence is posed after appendectomy and it is considered that there is no risk of metastases [5, 6]. The intermediate - size group of appendiceal nen> 1 cm but <2 cm is the group with a less clear situation . However, metastases also seem to occur very rarely in this subgroup, which comprises 525% of all appendiceal nen, their occurrence has been described in up to 10% in some series although the largest by moertel et al . Did not report any metastases in this subgroup . Right hemicolectomy is justified only in those rare tumours 12 cm in size but with positive or unclear margins or with deep mesoappendiceal invasion, higher proliferation rate (g2) and/or angioinvasion . However, in tumours larger than 2 cm, the risk of metastasis increases to 2540% [7, 8]. Tumours with a diameter> 2 cm should be treated by right hemicolectomy . In this study, the incidence of neuroendocrine neoplasm of the appendix, the epidemiological and histopathological features, the treatment that was used, and the clinical course were evaluated retrospectively . In total, 975 patients who were operated in ankara oncology education and research hospital between 2004 and 2013 with the diagnosis of acute appendicitis or who were operated for another reason but had incidental detection of a suspicious mass in the appendix and who underwent appendectomy were enrolled in this study . Among these patients, the record of the nine patients whose histopathological examination results showed appendiceal neuroendocrine neoplasm (according to the european neuroendocrine tumour society (enets) guidelines 2012) were examined retrospectively . Patients demographic characteristics, pathological characteristics (tumour size, localisation, invasion depth, and immunohistochemical staining characteristics), treatment, and time of following - up were analysed . The main referral cause of patients to the hospital was abdominal pain, nausea, and vomiting . Symptoms of heat intolerance, sweating, palpitations, and flushing were not detected . In a patient with left colon tumour neuroendocrine neoplasms were diagnosed in eight patients incidentally by appendectomy performed due to the prediagnosis of acute appendicitis, and in one patient by the examination of the appendectomy specimen that was taken because of the mass detection in the appendix during the surgery of the left colon tumour . The median age at diagnosis was 32 years (range, 1257 years) and the median tumour size was 0.6 cm (range, 0.21.5 cm). In all of the seven patients with tumour size equal to or smaller than 1 cm, the tumour showed the distal localisation, in one of the two patients with 12-cm tumour size, the tumour was in the proximal appendix, and in the other it was in distal appendix . When the relationship between tumour size and the depth of invasion of tumour considered in four of seven tumours was equal to or smaller than 1 cm, muscularis propria invasion was detected, in two patients there was serosa invasion, and in one patient there was deep mesoappendix invasion . Serosa invasion was detected in one of the two patients with 1 - 2 cm tumour size, and deep mesoappendix invasion in the other . Left hemicolectomy because of left colon tumour in one patient, right hemicolectomy was also applied for a suspicious appendix tumour . There was no lymph node involvement or residual tumour of the colon in the specimens of these three patients . Adjuvant chemotherapy was given to colon cancer patients, and the other patients were just followed . Patients were followed for a median of 78 months (range 6102 months). In the patient operated for colon tumour, metachronous colon tumour developed later . Gastrointestinal neuroendocrine tumours (traditionally referred to as carcinoid tumours) are seen most commonly in the small intestine with the frequency of 44.7%, and less frequently in the rectum (19.6%), appendix (16.7%), colon (10.6%), and stomach (7.2%). Appendiceal neuroendocrine neoplasms are diagnosed slightly more often in female than in male patients, at an average age of 4050 years [5, 10, 11]. In our series, median age was 34 years and the tumours were detected at a similar rate in both sexes and were seen with a frequency of 0.9% . In general, appendiceal nen are asymptomatic tumours seen in the preoperative period . In a study by robertson et al . The time between the formation of tumours and the beginning of symptoms was about nine years . Most of the time, acute appendicitis findings developing due to increased intraluminal pressure induced by the tumour clogging appendiceal lumen is the first and unique symptom . Carcinoid syndrome usually develops after liver metastases occur, and it is seen in 10% of all carcinoid tumours . Major findings are flushing, diarrhoea, skin manifestations (e.g. Pellagra), bronchospasm, and progressive congestive heart failure . Generally, since tumour size is small, the sensitivity of the imaging methods is not high . In our study group, except for the patient operated because of colon tumour, in all patients the cause of hospital referral was findings suggestive of acute appendicitis . Carcinoid syndrome was not seen in any patients . Only in one patient, abdominal tomography was performed preoperatively because of colon tumour without revealing a mass lesion in the appendix, and in this patient a mass was suspected during the surgery . 75% of cases, at the base of the appendix in 5%, and in the mid - section in 20% . In the literature, reported mean tumour size is 6 mm (range; 0.414 mm) [5, 14]. In eight patients, the tumour was at the tip of the appendix, and in one patient it was in the base of the appendix; the median tumour size was 6 mm in this study . Size of the appendiceal nen, localisation within the appendix, and the extent of invasion into the mesoappendix are essential criteria for the therapeutic and follow - up stratification of appendiceal nen, which are usually well differentiated (g1/2). While infiltration of the appendiceal serosa does not per se seem to be associated with poorer outcome, invasion into the mesoappendix shows a higher rate of lymphatic invasion than in cases without . The depth of invasion beyond 3 mm has been suggested as reflecting the aggressiveness of the disease . Additional criteria such as a ki67 index of 3% or higher (net - g2) or angioinvasion have been suggested as aiding with decision - making . Since diagnosis is often incidentally made after appendectomy, according to the characteristics of the tumour determined by pathology reports, sometimes second surgery may be required . In a series of 28 patients, all patient with nen were treated with right hemicolectomy . According to tumour size (1 cm, 12 cm, and> 2 cm) in specimens, if in these patients the operation decision was taken according only to ki-67 index, vascular invasion and extensive mesoappendiceal invasion without taking account tumour size, residual disease would be missed in 18% of appendiceal nen patients . In our series, right hemicolectomy was applied due to mesoappendix invasion detected in two patients, but residual malignancy was not found in the removed specimens in any of the patients . The prognosis for the majority of appendiceal nen is excellent, and the five - year survival of patients is more than 90% . However, the whole cohort including all tumour stages does not show such a favourable prognosis, with the five - year survival rate ranging between 70 and 85% . In cases with deep mesoappendiceal infiltration or angioinvasion, imaging as outlined above may be performed to rule out any residual disease . All other patients with larger tumours, metastases, or additional risk factors (r1 resection, tumour size> 2 cm), should be followed initially after 6 and 12 months postoperatively and then annually . Patients should be monitored by physical examination, 5-hiaa and chromogranin - a levels . Due to the possibility of synchronous or metachronous colorectal carcinoma development at more than 33%, screening with colonoscopy should be recommended to patients [3, 4, 18]. In our series, one patient developed metachronous colon tumour . The rate of the distant metastases in all of carcinoid tumours is 4%, and the most common sites of metastasis are the regional lymph nodes and liver . However, in a mayo clinic study, if removal of 90% of the metastasis is possible, palliative debulking surgery is recommended . In irresectable metastasis, hepatic artery ligation or embolization if there is irresectable metastasis, medical treatment (octreotide, interferon - alpha, anticancer agents such as capacitabine, dacarbazine, 5-fu, oxaliplatin, and temozolamide, bevacizumab targeting vegf, and everolimus targeting mtor) and radionuclide therapy should be planned in order to reduce the symptoms [2023]. In our series of patients, since carcinoid syndrome or metastasis was not detected, additional treatment was not applied to any of the patients . As a result, since preoperative diagnostic methods are inadequate, neuroendocrine neoplasms of the appendix are often diagnosed by pathological examination . In this disease, which has good long - term prognosis, if tumour size is smaller than 2 cm and there is no adverse prognostic factor, appendectomy is sufficient in the treatment . If tumour size is greater than 2 cm and there is tumour invasion beneath the serosa of the appendix or positive or unclear margins or with deep mesoappendiceal invasion, the presence of higher proliferation rate (g2), and/or angioinvasion after appendectomy, right hemicolectomy should be applied.
In publications dating back several decades, irregularities in the morphology of the femoroacetabular articulation have been implicated as a possible source of hip pain in young, active athletes . A french article published in 1979 described hip pain associated with structural abnormalities of the proximal femoral neck in athletes participating in hockey, football, soccer, rugby, martial arts, and tennis . More recently, developments pioneered by open hip surgeons have shown that morphologic abnormalities of both the femur and acetabulum underlie a large number of labral and chondral injuries in the hip [1, 29]. Wenger et al . Noted the presence of osseous abnormalities, including dysplasia and femoroacetabular impingement (fai), in the majority of patients with labral tears . This suggests that isolated treatment of soft tissue pathologies may not be adequate without concomitantly addressing underlying structural abnormalities . The concept of fai has been defined by ganz and colleagues [1, 8, 27]. In this condition, a structural or spatial abnormality of the femur (cam) or acetabulum (pincer) damages the chondrolabral structures during normal joint movement . The most common situation is a mixed cam and pincer pathology, occurring along the anterior femoral neck and the anterior flexion and internal rotation movements, abutment and impingement of the labrum and cartilage occurs . Sub - clinical slipped capital femoral epiphyses [15, 25], mal - union of femoral neck fractures, and decreased femoral anteversion are described causes of cam impingement . Relative posterior opening of the acetabulum (acetabular retroversion) or global (coxa profunda) overcoverage of the femur by the acetabulum are described causes of pincer impingement [23, 24]. Cam and pincer lesions lead to distinct patterns of labral and chondral damage [1, 10] and long - standing impingement is likely a significant cause of previously described idiopathic hip joint degeneration . Ganz et al . Developed an open surgical dislocation approach to decompress fai [7, 13]. However, we believe that an arthroscopic approach involves less post - operative morbidity and allows patients, including professional athletes, to return to high - functioning lifestyles . The purpose of this study was to define associated pathologies and determine if professional athletes could return to high - level athletics following arthroscopic decompression of fai . A retrospective chart review was performed of all professional athletes presenting for arthroscopic treatment of fai by the senior author between october 2000 and september 2005 . All athletes presented with debilitating hip pain and an inability to participate in their sport . All were diagnosed with fai based on physical and radiographic examination . Physical examination criteria included a positive impingement test or flexion abduction external rotation (faber) test . A positive impingement test was defined as groin pain with 90 degrees of hip flexion and maximal internal rotation . A positive faber test was defined as asymmetry in the distance between the lateral knee and the exam table between the injured hip and the non - injured hip . Radiographic criteria included decreased anterior and superior femoral head neck offset, acetabular retroversion (as defined by a positive cross - over sign), or coxa profunda (as defined by the medial acetabular border overlapping the ilioischial line). For these athletes with documented physical and radiographic evidence of fai, conservative treatment was limited to only 6 weeks . Based on evidence described by beck and colleagues, our thought was that conservative treatment could not address the underlying bony abnormalities and any further treatment delay in these highly active patients would likely precipitate further irreversible damage to the articular cartilage . Inclusion criteria for this study included professional athletes with at least one positive physical exam finding, at least one positive radiographic finding, and failure of at least 6 weeks of conservative therapy . Forty - five professional athletes from various sports met the inclusion criteria and were included in the study (table 1). Table 1pre - operative sports activities of the 45 professional athletessportno . Of patientshockey24golf6football5soccer3dance2baseball2martial arts1tennis1jockey1total45 pre - operative sports activities of the 45 professional athletes two portals (anterior, anterolateral) were established and a third portal (distal lateral accessory) was established as needed . At the time of arthroscopy, pincer lesions were typically identified in the superior acetabular quadrant . For the treatment of small pincer lesions (<2 to 3 mm), osteoplasty of the proximal femoral neck was occasionally sufficient to relieve the impingement . Patients with moderate or large - sized lesions underwent acetabular rim trimming, using an arthroscopic osteotome or 5.5 mm round motorized burr . Acetabular labral tears were arthroscopically identified, and based on previous research on the vascularity of the labrum [9, 11], detached and peripheral midsubstance tears were typically repaired [12, 22]. Care was taken to preserve as much healthy, viable labrum as possible, however, degenerative or frayed tissue was debrided to a stable remnant . Labral repair was performed with suture anchors to repair detached labral tears or to re - fix the labrum following iatrogenic detachment for complete resection of pincer lesions . The previously described technique involved placement of the anchors high on the acetabular rim in the area of detachment . Typically, one bioabsorbable anchor (bioraptor, smith+nephew, andover, ma, usa) was placed at the 12:00 acetabular position, and re - inforcement was placed either anteriorly or posteriorly of this area as needed . One limb of suture (ultrabraid, smith+nephew, andover, ma, usa) was passed between the labral tissue and the rim, and was retrieved through the substance of the labral tissue . In cases of inadequate availability of labral substance or in patients with highly degenerative, friable tissue, for midsubstance labral tears, a suture passer looped an 0-vicryl around the torn tissue to approximate the edges of the midsubstance split . The condition of the articular cartilage was evaluated according to the outerbridge classification system . For this study, the outerbridge classification system was defined as follows: grade 0, normal cartilage; grade 1, cartilage with softening and swelling; grade 2, a partial thickness defect with fissures on the surface that do not reach the subchondral bone or exceed 1.5 cm in diameter; grade 3, fissuring to the level of the subchondral bone in an area with a diameter more than 1.5 cm; and, grade iv, exposed subchondral bone . Chondral procedures performed in this group included chondroplasty and microfracture of lesions of the femoral head and acetabulum as needed . Following inspection of the central compartment, traction was released and the peripheral compartment was evaluated for cam lesions . A dynamic examination of the hip in all motion planes allowed for direct visualization of impingement at the femoroacetabular interface . For the treatment of cam lesions, an osteoplasty was performed with a 5.5 mm round motorized burr, restoring the anatomic concavity of the femoral head neck junction . Burring was performed from superior to inferior along the anterior femoral head neck junction, to a depth of approximately 57 mm and a width of approximately 812 mm . Care was taken to avoid resecting too distally along the femoral neck to avoid the lateral epiphyseal vessels . A final dynamic examination was performed to confirm adequate decompression, and a lack of entrapment of the labrum during joint motion . In general, the above - described procedure took approximately 23 h, depending on the degree of labral and chondral pathology . Traction time was limited to less than 2 h to reduce the risk of neurovascular compromise . Post - operative medical prophylaxis included an aspirin or a low - molecular weight heparin, a non - steroidal anti - inflammatory, and an antibiotic . Each of the 45 athletes underwent post - operative rehabilitation according to the following generalized protocol that was devised based on personal experience . For basic procedures involving decompression of fai and labral treatment, weightbearing a brace was prescribed to be worn for 10 days to protect the hip and limit abduction and rotation . Continuous passive motion (cpm) was used to apply 090 degrees of hip flexion for up to 8 h per day for 4 weeks . Night boots were worn for ten post - operative days to limit rotation during sleep . If capsular modification procedures (thermal capsulorrhaphy or capsular plication) were performed, rotation precautions were extended for a total of 21 days to avoid capsular stress . If microfracture was performed, weightbearing restrictions and cpm use were extended for a total of 8 weeks . It is our belief, based on clinical observation, that this, in addition to an early emphasis on passive hip motion (particularly internal rotation) reduces the incidence of adhesion formation . Active hip flexion was limited for 4 weeks, based on clinical observation, to minimize the risk of hip flexor tendonitis . Athletes were typically allowed to return to full competitive activity between 12 and 16 post - operative weeks . Return - to - play statistics were determined by retrospective chart review and personal follow - up communication with the athletes . A retrospective chart review was performed of all professional athletes presenting for arthroscopic treatment of fai by the senior author between october 2000 and september 2005 . All athletes presented with debilitating hip pain and an inability to participate in their sport . All were diagnosed with fai based on physical and radiographic examination . Physical examination criteria included a positive impingement test or flexion abduction external rotation (faber) test . A positive impingement test was defined as groin pain with 90 degrees of hip flexion and maximal internal rotation . A positive faber test was defined as asymmetry in the distance between the lateral knee and the exam table between the injured hip and the non - injured hip . Radiographic criteria included decreased anterior and superior femoral head neck offset, acetabular retroversion (as defined by a positive cross - over sign), or coxa profunda (as defined by the medial acetabular border overlapping the ilioischial line). For these athletes with documented physical and radiographic evidence of fai, conservative treatment was limited to only 6 weeks . Based on evidence described by beck and colleagues, our thought was that conservative treatment could not address the underlying bony abnormalities and any further treatment delay in these highly active patients would likely precipitate further irreversible damage to the articular cartilage . Inclusion criteria for this study included professional athletes with at least one positive physical exam finding, at least one positive radiographic finding, and failure of at least 6 weeks of conservative therapy . Forty - five professional athletes from various sports met the inclusion criteria and were included in the study (table 1). Table 1pre - operative sports activities of the 45 professional athletessportno . Of patientshockey24golf6football5soccer3dance2baseball2martial arts1tennis1jockey1total45 pre - operative sports activities of the 45 professional athletes two portals (anterior, anterolateral) were established and a third portal (distal lateral accessory) was established as needed . At the time of arthroscopy, pincer lesions were typically identified in the superior acetabular quadrant . For the treatment of small pincer lesions (<2 to 3 mm), osteoplasty of the proximal femoral neck was occasionally sufficient to relieve the impingement . Patients with moderate or large - sized lesions underwent acetabular rim trimming, using an arthroscopic osteotome or 5.5 mm round motorized burr . Acetabular labral tears were arthroscopically identified, and based on previous research on the vascularity of the labrum [9, 11], detached and peripheral midsubstance tears were typically repaired [12, 22]. Care was taken to preserve as much healthy, viable labrum as possible, however, degenerative or frayed tissue was debrided to a stable remnant . Labral repair was performed with suture anchors to repair detached labral tears or to re - fix the labrum following iatrogenic detachment for complete resection of pincer lesions . The previously described technique involved placement of the anchors high on the acetabular rim in the area of detachment . Typically, one bioabsorbable anchor (bioraptor, smith+nephew, andover, ma, usa) was placed at the 12:00 acetabular position, and re - inforcement was placed either anteriorly or posteriorly of this area as needed . One limb of suture (ultrabraid, smith+nephew, andover, ma, usa) was passed between the labral tissue and the rim, and was retrieved through the substance of the labral tissue . In cases of inadequate availability of labral substance or in patients with highly degenerative, friable tissue, standard arthroscopic knots fixed the repair to the rim . For midsubstance labral tears, a suture passer looped an 0-vicryl around the torn tissue to approximate the edges of the midsubstance split . The condition of the articular cartilage was evaluated according to the outerbridge classification system . For this study, the outerbridge classification system was defined as follows: grade 0, normal cartilage; grade 1, cartilage with softening and swelling; grade 2, a partial thickness defect with fissures on the surface that do not reach the subchondral bone or exceed 1.5 cm in diameter; grade 3, fissuring to the level of the subchondral bone in an area with a diameter more than 1.5 cm; and, grade iv, exposed subchondral bone . Chondral procedures performed in this group included chondroplasty and microfracture of lesions of the femoral head and acetabulum as needed . Following inspection of the central compartment, traction was released and the peripheral compartment was evaluated for cam lesions . A dynamic examination of the hip in all motion planes allowed for direct visualization of impingement at the femoroacetabular interface . For the treatment of cam lesions, an osteoplasty was performed with a 5.5 mm round motorized burr, restoring the anatomic concavity of the femoral head neck junction . Burring was performed from superior to inferior along the anterior femoral head neck junction, to a depth of approximately 57 mm and a width of approximately 812 mm . Care was taken to avoid resecting too distally along the femoral neck to avoid the lateral epiphyseal vessels . A final dynamic examination was performed to confirm adequate decompression, and a lack of entrapment of the labrum during joint motion . In general, the above - described procedure took approximately 23 h, depending on the degree of labral and chondral pathology . Traction time was limited to less than 2 h to reduce the risk of neurovascular compromise . Post - operative medical prophylaxis included an aspirin or a low - molecular weight heparin, a non - steroidal anti - inflammatory, and an antibiotic . Each of the 45 athletes underwent post - operative rehabilitation according to the following generalized protocol that was devised based on personal experience . For basic procedures involving decompression of fai and labral treatment, weightbearing a brace was prescribed to be worn for 10 days to protect the hip and limit abduction and rotation . Continuous passive motion (cpm) was used to apply 090 degrees of hip flexion for up to 8 h per day for 4 weeks . Night boots were worn for ten post - operative days to limit rotation during sleep . If capsular modification procedures (thermal capsulorrhaphy or capsular plication) were performed, rotation precautions were extended for a total of 21 days to avoid capsular stress . If microfracture was performed, weightbearing restrictions and cpm use were extended for a total of 8 weeks . It is our belief, based on clinical observation, that this, in addition to an early emphasis on passive hip motion (particularly internal rotation) reduces the incidence of adhesion formation . Active hip flexion was limited for 4 weeks, based on clinical observation, to minimize the risk of hip flexor tendonitis . Athletes were typically allowed to return to full competitive activity between 12 and 16 post - operative weeks . Return - to - play statistics were determined by retrospective chart review and personal follow - up communication with the athletes . The average age of the athletes at the time of surgery was 31 years (range: 1761). Eleven athletes (24%) previously underwent hip arthroscopy by multiple primary surgeons for the treatment of labral and chondral pathologies and experienced a recurrence of hip symptoms . The average time to follow - up was 1.6 years (range: 6 months to 5.5 years). Twenty - two athletes (49%) were treated for an isolated cam lesion and three athletes (7%) were treated for an isolated pincer lesion . Twenty - one athletes (47%) had a mixed pathology of both cam and pincer lesions . Twenty - five patients (56%) underwent either labral repair or re - fixation following rim trimming with suture anchors (average 1.3 anchors per patient, range: 13). Five patients had labral debridement; one patient had a detached tear, one had frayed labral tissue, and three patients had complex tears consisting of detached, frayed, and flap components . Twenty - one patients (47%) had a grade iv acetabular chondral defect; 14 underwent arthroscopic microfracture, 5 underwent thermal chondroplasty, and 2 patients had no treatment due to the diffuseness of their disease . Three patients (7%) had a grade iv femoral head chondral defect; one was treated with microfracture, one with chondroplasty, and one patient had no treatment due to the diffuseness of his disease . Iii acetabular chondral defect (13 treated with chondroplasty and 1 treated with microfracture) and 11 (24%) had a grade i iii femoral head chondral defect (all 11 were treated with chondroplasty). Four patients (one baseball player, one football player, one hockey player and 1 golfer) had extensive diffuse oa at the time of arthroscopy, but opted to delay arthroplasty . Twenty - six patients (58%) had a partial tear of the ligamentum teres and 3 patients (7%) had a complete ligamentum teres avulsion . Thirteen (29%) patients underwent thermal capsulorrhaphy and 9 (20%) underwent plication for capsular redundancy . Seventeen patients (38%) had loose bodies in the joint and two patients required excision of myositis ossificans of the rectus femoris . Three patients (7%) underwent cheilectomy of a stenotic cotyloid fossa and 9 patients (20%) underwent arthroscopic release of a tight iliopsoas . Figure 1 illustrates restoration of femoral head neck concavity in an athlete treated for cam impingement (fig . 1) and fig . 2 illustrates successful removal of the anterior acetabular overhang in an athlete treated for pincer impingement (fig . 2). 1a 27-year - old professional hockey player was evaluated for right hip pain . A a pre - operative cross - table lateral radiograph demonstrated convexity at the anterior femoral head neck junction . B the presence of a cam lesion was verified arthroscopically . The femoral head (fh) and acetabulum (ac) c a motorized burr was used to restore anterior concavity at the head neck junction . This was visualized at the superior acetabular (ac) position (12:00) with the camera in the anterior portal . D a post - operative film verified successful decompression of the cam impingement and restoration of the femoral head neck junction concavityfig . A a pre - operative ap radiograph demonstrated a cross - over sign of the right acetabulum, indicating acetabular retroversion . B the presence of a pincer lesion was verified arthroscopically with the camera in the anterolateral portal with excessive bone along the anterior c a motorized burr in the anterior portal resected the lesion with the burr shield placed against the labrum (l) for protection . E a post - operative film verified the successful removal of pincer impingement and lack of retroversion in the superior region of the acetabulum a 27-year - old professional hockey player was evaluated for right hip pain . A a pre - operative cross - table lateral radiograph demonstrated convexity at the anterior femoral head neck junction . The femoral head (fh) and acetabulum (ac) are visualized in the peripheral hip compartment with the camera in the anterior portal . C a motorized burr was used to restore anterior concavity at the head neck junction . This was visualized at the superior acetabular (ac) position (12:00) with the camera in the anterior portal . D a post - operative film verified successful decompression of the cam impingement and restoration of the femoral head neck junction concavity a 22-year - old professional football player was evaluated for right hip pain . A a pre - operative ap radiograph demonstrated a cross - over sign of the right acetabulum, indicating acetabular retroversion . B the presence of a pincer lesion was verified arthroscopically with the camera in the anterolateral portal with excessive bone along the anterior superior acetabulum (ac). C a motorized burr in the anterior portal resected the lesion with the burr shield placed against the labrum (l) for protection . E a post - operative film verified the successful removal of pincer impingement and lack of retroversion in the superior region of the acetabulum forty - two athletes (93%) returned to professional sport following hip arthroscopy . Three players (1 football player, 1 hockey player, and 1 baseball player) did not return to play following arthroscopy . Five athletes (11%) required re - operation . Three underwent lysis of adhesions and two had symptomatic treatment of extensive osteoarthritis . All of the patients who underwent revision surgery for lysis of adhesions returned to professional play and the two with extensive osteoarthritis did not return to play . Thirty - five of the 45 athletes (78%) remained active at the professional level at an average of 1.6 years after hip arthroscopy . Intra - articular hip injuries are a recognized cause of pain, mechanical symptoms, and disability in athletes . Traumatic intra - articular injury results from acute injury, including hyperabduction injuries, direct hip contact, and joint dislocation or subluxation . Various motions exerted during sport, particularly flexion combined with internal rotation (hockey - goalie stance), have been suggested as potential causes of overuse hip injury [19, 21]. In this position, a cam lesion extending anteriorly or superiorly from the proximal femoral neck would impinge at the anterosuperior acetabular rim . Combining this understanding with recent reports by wenger et al . And ganz et al . [1, 2, 8, 13], morphological abnormalities of the femoroacetabular joint ganz and colleagues described fai resulting from structural abnormalities of the proximal femoral neck (cam), acetabular rim (pincer), or most commonly, a combination of the two pathologies (mixed). Damage to the articular cartilage and acetabular labrum results from this pathologic bony contact and fai is a likely trigger of early hip joint degeneration . In our experience, fai is a common problem that has only recently been recognized in athletes with a primary hip complaint . However, increasing recognition of fai in a high - demand, competitive athletic population encouraged the development of a less invasive arthroscopic approach to facilitate prompt return to competitive play . Various studies have demonstrated that hip arthroscopy is a safe and effective intervention in athletes with intra - articular injuries [4, 17, 19, 20]. Philippon reported on a series of ten elite athletes who underwent hip arthroscopy for labral debridement with thermal capsulorrhaphy . Mccarthy et al . Reported 80% excellent results following hip arthroscopy in elite athletes (average follow - up of 23.6 months). Byrd et al . Reported a series of 44 hip arthroscopies in 42 recreational, high school, collegiate, elite, and professional athletes . Post - operative improvement, as quantified by the modified harris hip score, was present in all classes of athletes and in athletes undergoing any arthroscopic procedure (removal of loose bodies, debridement of ligamentum teres, excision of osteophyte, labral excision, microfracture, chondroplasty). Byrd et al . Reported better results in athletes who recalled a traumatic onset to their hip symptoms, when compared to those with an acute or insidious onset . The authors suggested that an unaddressed pre - disposition to injury might have had a negative impact on self - reported outcomes in athletes . Fai may be the unaddressed pre - disposition to which byrd and colleagues were alluding . While these above articles demonstrated successful outcomes following debridement procedures in athletes, there are a few caveats that must be addressed . First, it is likely that simple excision of damaged or diseased tissue, like with meniscal tears or loose bodies, can provide short - term abatement of pain and mechanical symptoms, and allow an athlete to return to professional athletics . However, recent studies have shown that simple hip debridement procedures failed to address the primary pathology in a vast majority of cases [1, 29]. A recent unpublished study performed by the senior author showed that a major cause of revision hip arthroscopy in all patients was treatment of labral and chondral re - injury and decompression of previously unaddressed fai . Future studies examining the clinical and radiographic long - term outcomes of athletes treated with debridement procedures need to be compared to those athletes treated for fai at an early stage of this disease . The purpose of this study was to simply state that despite the fact that fai surgery is a more extensive procedure than debridement, professional athletes can still return to play following this procedure . An arthroscopic approach to treat fai was recently reported; however, early outcome studies are lacking . The results of this study show that professional athletes with fai can return to high - level competitive sport following this procedure . Three athletes did not return to sport (1 hockey player, 1 football player, and 1 baseball player), however all had diffuse hip osteoarthritis present at the time of arthroscopy . One patient (a senior pga golfer) was able to return to sport following arthroscopy, despite his diffuse osteoarthritis . Based on this information, it seems that arthroscopic treatment of fai in the presence of oa can allow patients to return to low - impact, however, not likely high - impact professional sports . A larger cohort of patients is needed to test this hypothesis . A recent study showed that up to 30% of the femoral neck may be resected before it is structurally compromised . In this series of patients, additional potential complications include adhesion formation at either the capsulolabral margin or between the capsule and the site of bony resection . In this series of 45 athletes, 3 patients (7%) required revision arthroscopy for the lysis of adhesions (loa). Two additional athletes had extensive oa at the time of primary arthroscopy and subsequently underwent revision arthroscopy for symptomatic treatment of oa, including osteophyte debridement . There were no reported cases of heterotopic ossification or neurovascular compromise, including lateral femoral cutaneous, sciatic, and pudendal neuropraxia, reported, although this statistic may have been underestimated due to the nature of this professional athlete cohort . A limitation of this study was the inherent selection bias involved with the study of professional athletes . This patient sub - group was financially motivated to return to play and may have been less likely to report post - operative symptoms and complications than the rest of patients treated for fai . Despite this, we believed that a return - to - play analysis was critical to assess the outcomes of fai treatment in a high - demand population . This cohort of professional athletes was debilitated prior to hip arthroscopy, and following intervention, was able to perform in physically intense professional sport activities . Also, the athletes precise reason for retirement and whether it was related to their hip injuries was difficult to discern . For this reason, given the relatively short span of all professional athletes careers, we chose to include all athletes at a minimum of 6 months post - operative . This time period was selected as an ample time period for completion of post - operative rehabilitation and return to sport . Another limitation of this study was the lack of follow - up subjective and objective data . Current outcomes instruments (modified harris hip score and the non - arthritic hip score) have not been validated for use in high - level athletes . It is our belief that these scoring systems fail to address the activities that are most limiting to athletes, and hence, underestimate the degree of debilitation in professional athletes . Evaluating the applicability of current scoring systems in athletes, and developing new outcomes instruments for athletes will be the focus of future studies . Other limitations of this study included unequal distribution of gender (42 males and 3 females) and professional sports . Because different sports place different demands on the hip, we cannot draw conclusions among the various sports . The arthroscopic treatment of fai represents the evolution of hip arthroscopy . In the past, recent developments, particularly by ganz and colleagues, have enlightened us to the pathology of fai and the associated treatment options . In order to treat high - demand patients with fai this study has demonstrated that full return to professional competitive sport is possible following arthroscopic treatment of fai . Additional studies are needed to determine the effect on long - term joint degeneration of early surgical intervention to treat fai.
Type 2 diabetes (t2 dm) and obesity have become epidemic in malaysia . According to the latest national health and morbidity surveys, 14.9% of malaysian adults aged 30 years and above are diabetic and they are often obese or overweight [1, 2]. Moreover, diabetic nephropathy (dn) is the most common cause of end - stage renal disease (esrd) and contributes to 57% of patients with t2 dm in this country . Although t2 dm represents a preventable and treatable cause of esrd, the number of esrd cases caused by t2 dm has increased and accounts for more than 50% of incident dialysis patients [35]. Pentraxin 3 (ptx3) is an acute - phase glycoprotein and a soluble receptor acting as an opsonin . Thereby, its levels may reflect more directly the inflammatory status of the vasculature [6, 7]. Recently, several clinical investigations have demonstrated that elevated plasma ptx3 levels are associated with cardiovascular [8, 9] and chronic kidney diseases (ckd) [10, 11]. Furthermore, plasma ptx3 levels are inversely associated with body mass index (bmi) suggesting that ptx3 may play a role in obesity and metabolic syndrome [12, 13]. Interestingly, with the approach of genome - wide scan and linkage analysis, chromosome 3q is found to be linked with diabetes and dn in many ethnic groups [1416]. The gene encoding for ptx3 protein is located in chromosome 3q25.32 and resides in the linkage region . Have shown that ptx3 is positively associated with proteinuria in turkish subjects with t2 dm hypertensive patients, while renin angiotensin system blockade lowers plasma ptx3 levels in the patients [17, 18]. However, there are gender and racial differences of plasma ptx3 levels [7, 19], by which the association of ptx3 with kidney dysfunction may be influenced . In the present study, we examined plasma ptx3 levels in a malay cohort, including ngt subjects, t2 dm patients with and without dn . The aim of our study was to investigate the association of plasma ptx3 levels with t2 dm and dn in this malay population . Data from our study are also useful for better understanding the different effects of ptx3 in dn and ckd . We collected the samples of subjects with ngt and patients with t2 dm from the collaborating centers all over malaysia . The ethnic distribution of our study subjects was 67.6% malay, 15.3% indian, 14.8% chinese, and 2.3% indigenous sabahans and sarawakians . To avoid the error caused by ethnic stratification, indian, chinese, indigenous sabahans, and sarawakians were excluded in the present study . Finally, 103 (50 males/53 females) malay individuals with ngt (controls) and 193 (99/94) malay patients with t2 dm (cases) were included into the analyses . Diagnoses of t2 dm were done based on the world health organization (who) criteria . The diagnoses of dn were based on urine albumin - to - creatinine ratio (acr) suggested by ada . The patients with t2 dm and normoalbuminuria (acr <3.5 mg / mmol) were considered as controls for dn, while the patients with macroalbuminuria (acr 35 mg / mmol) and esrd who needed dialysis were included as the cases for dn . Except for the patients with esrd, all other subjects were required to give urine under fasting conditions early in the morning . Clinical characteristics of all malay subjects with ngt and t2 dm with and without dn are summarized in table 1 . Informed consent was obtained from all subjects, and the study was approved by the local ethical committees . Data and materials transfer agreement from the institute for medical research, malaysia to karolinska institutet, sweden was signed prior to the study . Body weight and height were measured using a calibrated digital scale (seca, birmingham, uk). The who / international association for the study of obesity (iaso)/international obesity task force (iotf) has proposed bmi cut - off values of 23.024.9 kg / m for classification of overweight and of 25.0 kg / m for obesity for adult asians . Based on the malaysian clinical practice guidelines, subjects with bmi value 23.0 kg / m are considered as overweight . Systolic and diastolic blood pressures were measured using a digital sphygmomanometer (omron healthcare, inc ., creatinine in serum and urine were measured using randox assayed multisera (randox laboratories ltd ., urine or serum was mixed with sodium hydroxide in biochemistry analyzer (selectra e). The amount of the complex formed was measured at wavelength of 490 to 510 nm . Estimate of glomerular filtration rate (gfr) in the dialysis patient was calculated by the mean of renal urea and creatinine clearance from a 24-hour urine correction . A total of 25 ml venous blood samples were collected from each subject early in the morning after an overnight fasting and then stored at 80c . Plasma ptx3 concentrations were determined using a commercial enzyme - linked immunosorbent assay kit (quantakine dptx 30; r&d systems inc ., briefly, 20 l of standard and plasma samples was assayed duplicate in the microtiter plate wells coated with a specific ptx3 monoclonal antibody followed by incubation at room temperature for 2 hours . Anti - ptx3 polyclonal antibody conjugated to alkaline phosphatase was added to each well and incubated for two hours at room temperature . After washing step, 200 l of substrate solution was added to each well followed by incubation for 30 minutes at room temperature . The solution of 2 n sulfuric acid was added to each well to stop the reaction . Absorbance was measured at 450 nm with corrections set at 540 nm using microplate reader . The values of plasma ptx3 levels were extrapolated from a curve drawn using a standard ptx3 . All data were expressed as mean (95% ci) for normally distributed variables and as geometric means (95% ci) for nonnormally distributed variables . Normal probability plots were created and parameter distributions were transformed to the common logarithm for obtaining a normal distribution before performing statistical analysis . The one - way analysis of variance (anova) was used for comparisons involving more than two groups or independent t - test for comparison between two groups . Pearson and spearman analyses were conducted to determine correlations with continuous and noncontinuous variables, respectively . All analyses were performed using pasw statistic base 18 (spssinc, chicago, usa). In both males and females, there was no difference in age, waist circumference, and hba1c between t2 dm patients and ngt subjects . Although plasma ptx3 levels in males and females differed significantly, ptx3 had no relationship with age in males (p = 0.647) or females (p = 0.626). Thus, all subsequent analyses of plasma ptx3 levels were done separately in males and females . Our analyses indicated that plasma ptx3 levels in t2 dm patients with and without dn were lower as compared with ngt subjects in males (2.62 versus 3.98 ng / ml; p = 0.021) but not in females (3.24 versus 3.09 ng / ml; p = 0.748). Figure 1(a) showed that plasma ptx3 levels were consistently decreased from ngt to t2 dm without dn and to the patients with dn in males (3.98, 2.78, and 1.63 ng / ml; p = 0.008 anova test). Among males, the patients with dn had lower ptx3 levels compared to t2 dm without dn (1.63 versus 3.08 ng / ml; p = 0.013). In females, however, there was no statistically significant difference of the mean values of plasma ptx3 levels among ngt and t2 dm with and without dn (3.09, 3.55, and 2.11 ng / ml; p = 0.262, anova test) (figure 1(b)). There was a negative correlation between plasma ptx3 levels and bmi in male subjects with ngt (r = 0.390; p = 0.012) (figure 2) but not in females (p = 0.330). The correlation between ptx3 and bmi was not found in all male and female t2 dm patients with and without dn . In malaysia, the adults with bmi value 23.0 kg / m are considered to be overweight . To further understand whether the association between ptx3 and dn in t2 dm was influenced by bmi, we performed the comparative analyses in the patients with overweight (bmi 23 kg / m) and lean patients (bmi <23 kg / m), respectively . In males with overweight, we found that plasma ptx3 levels were gradually decreased from subjects with ngt to t2 dm patients without dn and to the patients with dn (3.68, 2.60 and 1.42 ng / ml; p = 0.044, anova test) (figure 3). In lean males and also in all females, plasma ptx3 levels of ngt and t2 dm with and without dn were varied but not with any statistical significance . In the present study, we analyzed plasma ptx3 levels in malay subjects with ngt and t2 dm with and without dn . Lower levels of ptx3 were found to be associated with t2 dm and dn in males but not in females . Furthermore, ptx3 was found to be inversely associated with bmi in males with ngt . The correlation was not observed both in males and females with t2 dm and dn . Males have a higher prevalence of t2 dm and dn in many populations including malaysians [1, 35]. Epidemiologic reports have demonstrated that dn is 30% more frequent in males than in females . Genetic studies have also showed that dna polymorphisms in the genes of sex - determining region y - box 2, angiotensin ii type 1, and type 2 receptors are associated with dn with gender - specific effects [2628]. Previously, yamasaki et al . Observed that plasma ptx3 levels between males and females in a healthy japanese population are different . In the present study, we demonstrate that plasma ptx3 levels gradually decreased from ngt to t2 dm without dn to t2 dm with dn particularly among males but not in females . Furthermore, an inverse correlation between ptx3 and bmi was found in male subjects with ngt . This correlation was not seen in all females and males with t2 dm and dn . Taking together, data from previous and present studies implicate that ptx3 most likely has gender - specific effects in t2 dm and dn, which should be taken into our consideration in further investigations . Several studies have reported that increased ptx3 levels are associated with impaired renal function in ckd [10, 11]. The similar association of ptx3 with dn is seen in turkish patients with t2 dm . In the present study, however, we demonstrate that decreased ptx3 levels are associated with dn in malay men with t2 dm . Dubin et al . Have demonstrated that there are racial differences of ptx3 in term of association with kidney dysfunction . Second, the ages of turkish t2 dm subjects with dn (at 42 years old) are younger and their duration of diabetes are shorter compared to malay t2 dm patients with dn (at the age of 55 years old) in the present study . Furthermore, clinical observations have indicated that two main causes of ckd are diabetes and high blood pressure, which are responsible for up to two - thirds of the cases . The progresses and mechanisms of dn and ckd may be different, while ptx3 may have different effects in these two diseases . We have shown that plasma ptx3 levels were inversely correlated with bmi in males with ngt, which is consistent with previous reports [7, 12, 13]. In the present study, there is a limitation with lack of lean subjects because all patients with t2 dm and female subjects with ngt had mean values of bmi at least 27.2 kg / m . Recently, a study has demonstrated that ptx3 is expressed in adipose tissue, and its tissue specific expression reflects endothelial dysfunction . Although we did not analyze plasma levels of adiponectin in this malay cohort, the accumulated documents have shown that adiponectin is inversely proportional to obesity and t2 dm in different populations including malaysians . Plasma / serum adiponectin levels in the patients with t2 dm and obese subjects are decreased compared to that in healthy control subjects [31, 32]. Therefore, we hypothesize that ptx3, as similar to adiponectin, may have protective effects in increased body weight . Further investigation is needed to fully understand the cellular mechanism of ptx3 reduction in t2 dm and dn . In conclusion, the present study provides the first evidence that decreased plasma ptx3 levels are associated with t2 dm and dn in malay men and also suggests that ptx3 may have different effects in dn and ckd.
One of the methods of partial ureter substitution is the use of free bladder autologous mucosa flaps . This method has been in use for about 50 years . Despite previous reports describing the benefits of this method, in reconstructive surgery, up to now, it has not been used in clinical practice . Our poor morphologic results with the use this method prompted us to perform microangiography of the newly constructed part of the ureter with this method to definitively resolve the problem of neovascularization in this area . The experiments were carried out on 29 mongrel dogs, as previously described [14]. All animals were operated on in the same manner (figure 1), with general endotracheal anesthesia . After grafting, four of the experimental animals were additionally subjected to perfusion of the vascular bed with a radiopaque medium called micropaque (barium sulphuricum) (nichols roche, france, distributed by schering germany). This was done to ascertain neovascularization in the supplemented part of the ureter by a tube constructed from the free mucosa flap of the urinary bladder . Microangiography was performed after a longitudinal median laparotomy of 34 cm over the kidney vessels . The aorta was ligated and a cannula connected with the perfusion set (figure 2) was inserted into the aorta . To avoid errors in performing the operation and interpreting our results, we carefully studied the literature [512] and consulted with other investigators about the principles of this kind of experiment . The perfusion of 500 ml 30% micropaque (barium sulphuricum) (nichols roche, france, distributed by schering germany), with 0.9% nacl was transfused and subsequently 30% micropaque (barium sulphuricum) (nichols roche, france, distributed by schering germany) with 10% buffered formalin . Perfusion was performed under constant pressure of 140/80 mm hg, at the temperature of 37c, for 30 min . After the procedure, all the experimental animals were euthanized and the samples were harvested and stored for 6 h at 20c . Then, a segment of ureter measuring 10 cm in length was excised (5 cm above and 5 cm below the graft suture site), showing the surface vessels filled with micropaque (figure 3). At the end of the procedure, the specimen was stitched on the celluloid plate and harvested with 10% formalin, buffered to 7.6 ph . After 14 consecutive days, the specimens were irradiated to show the visible net of the new vascular bed (figure 4). Then, they were cut into 2-mm - thick slices using a microtome and were irradiated by direct exposure and visualized on celluloid film . Exposure time was 10 min, electric voltage 20 kv, and current intensity 5 ma . We used single - sided celluloid film type tn-12, (bydgoszcz manufacturing fotochemic plant, poland), which allowed us to obtain image of capillaries of up to 100200, because these celluloid films contain a one - sided photosensitive layer . A stepwise process of the ureter substitutions from 1 dog is shown in figures 13 . Microscopic evaluations showed the complete regeneration of the mucosa of the supplemented ureteral segment [14]. The defects in the ureters were not bridged by the smooth muscle [14]. The experimental findings showed a massive periureteral fibrosis, due to reparative or reactive process, which was considered as the main reason for the failure of our interventions [14]. One of the most common causes of scarring or stricture of the ureters is ureter surgery . The reason for this is that the arteries going to the uterus are very close to the ureters . During surgery, the amount of scarring and inflammation that occurs after surgery can be very dense and as a consequence leads to hydronephrosis and destroying the kidney . Previous reports described the benefit of free autologous bladder mucosa graft for the reconstruction of the ureter . However, unpublished observations have generated a significant interest and a need to re - evaluate autologous ureter grafts constructed from the free autologous mucosa flap; therefore, this method has not been used in clinical practice . It should be noted that the results of our previous studies [14] and of the present study do not support a free bladder mucosa flap as a viable clinical therapy for ureteral reconstruction, in spite of the positive reports of the above - mentioned authors . The present animal study suggests that the surgical ureteral reconstruction using this method or grafted tissue is not suitable to achieve clinically desirable results.
Total body irradiation (tbi) is a radiotherapy technique mostly used as a part of the hematopoietic stem cell transplantation (hsct). The role of tbi in conjunction with chemotherapeutic agents is to destroy malignant or genetically disordered cells and to immunosuppress the patient prior to hsct . The immune system of the patients has to be suppressed prior to hsct, which otherwise could reject the donor cells . Among the various modalities of patient preparation prior to bone marrow transplantation, tbi is advantageous compared to other methods for achieving a homogeneous dose delivery and for boosting or shielding the dose to any specific sites if required . Tbi is a complex treatment procedure and differs from routine radiotherapy treatment techniques inpatient setup, treatment distance, and field size . Hence, it is mandatory to monitor the accurate delivery of the prescribed dose during tbi through in vivo dosimetry checks . Dosimetry for tbi is generally performed using silicon diodes or thermoluminescent dosimeters (tlds). [36] accurate results can be obtained with lithium fluoride (lif) tld chips as their dependence on dose rate, direction, energy of the radiation beam, and temperature is less significant compared to the other available dosimeters like ionization chamber and semiconductor devices . It is recommended to analyze the in vivo dosimetry results carried out on a number of patients to check the efficiency of the technique being used . Though there have been a number of publications available on tbi in vivo dosimetry, the treatment technique used was not entirely similar among these studies . Most of the studies have used bilateral treatment fields with the patient lying in the supine position . Published results available on tbi in vivo dosimetry in the technique using anterior to posterior and posterior to anterior (ap pa) treatment fields with the patient lying on the lateral decubitus position are rare . The objective of this study is to report and analyze the results of our in vivo dosimetry checks measured with tld chips on tbi using ap pa treatment fields . The choice of a technique in a particular hospital depends on many factors like available equipment, photon beam energy, maximum possible field size, patient dimension, and treatment distance . Specific treatment parameters to be determined include field size, collimator angle, treatment distance, dose per fraction, dose rate, total dose, number of fractions per day, interval between fractions, beam energy, geometry to achieve dose homogeneity, bolus or beam spoilers to increase skin dose, shielding, and dose compensation requirements (e.g., lungs, kidneys). In our hospital, ap pa technique with 6 mv photon beam from linear accelerator (linac), oncor expression (ms . Vacuum cushion was used to keep the patient immobilized in the treatment position during treatment execution . Patient was made to lie on one side in the vacuum cushion at a source to surface distance (ssd) of 400 cm . Collimator jaws were opened to a maximum field size of 40 40 cm, with the collimator rotated to 45 so as to fully cover the patient body . Patients were asked to keep both arms across the chest and their fist over lung region of the chest to compensate for the tissue inhomogeneity in the lung . No separate blocks were used to shield lung, as it was difficult to introduce and match the blocks over lungs in the tbi technique with the patient lying on lateral decubitus position . A beam spoiler made of polymethylmethaacrylate (pmma) of thickness 2 cm was introduced between the patient and the machine at a distance of around 50 cm from the patient surface to avoid the skin - sparing effect . Total prescribed dose of 12 gy (150 cgy per fraction) was delivered to the patients over eight fractions of treatment in 4 days . Each day, two fractions of treatment were given with a gap of minimum 6 h. treatment was delivered at a dose rate of 6 cgy / min at the treatment distance by setting 100 monitor units (mu)/min in linac . Number of mus required to deliver the per field prescription dose of 75 cgy was in the range of 13001400 mus depending on the average thickness of the patient . During patient preparation, anterior posterior (a - p) thickness was measured at forehead, neck, chest, abdomen, pelvis, thigh, knee, and ankle to calculate the average thickness of the patient . Test dose of 20 cgy (10 cgy from anterior field and 10 cgy from posterior field) was delivered to the patients prior to treatment for dosimetric verification . Tld chips are the standard detector used for patient in vivo dosimetry in our tbi treatments . In vivo dosimetry was carried out by pasting tld chips at multiple sites throughout the patient body to measure the dose received . After ensuring acceptable dose results from the test dose measurements, patients were taken up for further treatment . Radiation output and depth dose profiles of the linac measured at the tbi setup were used for the mu calculation . Percentage depth dose (pdd) values measured for standard 100 cm ssd and converted for 400 cm ssd using the mayneord factor differ from the actual pdd for tbi setup, as the difference in ssd is very large . Hence, it is recommended that a direct measurement of the pdd and output should be obtained at the tbi setup . Rfa-300 radiation field analyzer (scanditronix wellhofer, uppsala, sweden) having mylar window on one wall was used to measure the pdd at the tbi setup . High - doped p - type silicon diodes (hi - p si) were used as the detectors . The reference detector was also kept beyond the beam spoiler (close to the phantom) in the same spectrum of beam energy . Accuracy of pdd data measured using diode was verified using gafchromic films at multiple depths (absolute measurement). Pdd values generated using mayneord factor for 400 cm ssd at every 5 mm were compared with the pdd values measured for tbi setup [figure 1]. Also, it was observed that for the pdd measured at tbi, the depth of dose maximum (dmax) had shifted to 4 mm depth compared to 15 mm depth for the pdd generated using mayneord factor . Based on these observations, the bolus material to keep the tld during in vivo dosimetry was made of 5 mm thickness to achieve buildup . Comparison of pdd curve obtained at tbi setup and pdd curve obtained at 100 cm ssd and converted using mayneord factor output measurement of the linac at the tbi distance was measured using solid water slabs (density: 1.03 g / cc) of dimensions 30 cm 30 cm . Multiple solid water slabs were used to make a phantom of 20 cm thickness . A 0.6 cc cylindrical ionization chamber (ptw, freiburg, germany) with unidos care was taken to reduce the direct exposure to dosimetry cable to minimize the leakage . The ionization chamber was kept at a depth of 10 cm and irradiated with 500 mu . The meter reading obtained was used to calculate the radiation output using the pdd value obtained at 10 cm depth for tbi setup . Iaea trs-398 formalism for output measurement of high - energy photon beams was used to calculate the output taking into account the ion recombination, polarity correction, and temperature and pressure correction . Two sets of lif tld-100 chips (harshaw, solon, oh, usa) of dimension 3.2 mm 3.2 mm 1 mm were used in this study . Tld chips were annealed at 400c for 1 h using fisher scientific thermal furnace, followed by gradual cooling at room temperature . A semi - automatic tld reader (harshaw 3500) was used to read the response from the tlds . Prior to start using the tld chips for dosimetry, both sets of tld chips were calibrated in order to establish dose as a function of tld reading . All the tlds were then annealed and irradiated with 6 mv photon at 5 cm depth in solid water phantom (density: 1.03 g / cc) with a 10 cm 10 cm jaw opening in the linac at 100 cm ssd to a dose of 200 cgy . During tld calibration and tbi in vivo dosimetry tld chips showing response within 1% of the mean response of the entire set were chosen as golden chips . After identifying the golden chips, tld chips were again annealed and irradiated in the standard setup to a known dose . The reader response to these golden chips was used as the calibration factor for the tld reader . The remaining tld chips other than the golden chips, known as field dosimeters, were read to generate their unique calibration factors . These field dosimeters were then used as the detectors in tbi in vivo dosimetry . A glow curve image obtained from the tld reader during tld calibration dosimetry results of 20 tbi patients (aged between 3 years and 28 years) treated between march 2008 and december 2011 were analyzed in this study . The intention of the in vivo dosimetry check was to ensure that the prescription dose was delivered accurately and uniformly throughout the patient's body . Hence, before using tld for every tbi patient, both sets of tlds were exposed to a known dose (200 cgy) at the standard setup used for tld calibration . Subsequently, only those tlds whose response fell within 3% of the given dose during this sensitivity check were used for the in vivo dosimetry . Tlds were recalibrated if the whole set of tlds showed more than 3% difference between the given dose and the measured dose during this sensitivity check . The measured dose values by each tld set during the multiple sensitivity checks performed were averaged out and are shown in figure 3 . Average of the measured doses by each tld during the sensitivity tests tlds along with wax bolus over each tld were kept at forehead, mediastinum, chest, abdomen, pelvis, thigh, and calf during in vivo dosimetry . Thickness of the bolus was maintained as 5 mm to achieve buildup region . From the results, minimum, maximum, and average measured dose values at each anatomical site prescription dose was divided into different ranges (<95%, 95105%, and> 105%), and an analysis was made for all sites on how many number of times the measured dose falls in each dose range . Mean dose to the whole body with standard deviation was calculated for each fraction of the treatment to check the homogeneity in the dose delivery . The choice of a technique in a particular hospital depends on many factors like available equipment, photon beam energy, maximum possible field size, patient dimension, and treatment distance . Specific treatment parameters to be determined include field size, collimator angle, treatment distance, dose per fraction, dose rate, total dose, number of fractions per day, interval between fractions, beam energy, geometry to achieve dose homogeneity, bolus or beam spoilers to increase skin dose, shielding, and dose compensation requirements (e.g., lungs, kidneys). In our hospital, ap pa technique with 6 mv photon beam from linear accelerator (linac), oncor expression (ms . Vacuum cushion was used to keep the patient immobilized in the treatment position during treatment execution . Patient was made to lie on one side in the vacuum cushion at a source to surface distance (ssd) of 400 cm . Collimator jaws were opened to a maximum field size of 40 40 cm, with the collimator rotated to 45 so as to fully cover the patient body . Patients were asked to keep both arms across the chest and their fist over lung region of the chest to compensate for the tissue inhomogeneity in the lung . No separate blocks were used to shield lung, as it was difficult to introduce and match the blocks over lungs in the tbi technique with the patient lying on lateral decubitus position . A beam spoiler made of polymethylmethaacrylate (pmma) of thickness 2 cm was introduced between the patient and the machine at a distance of around 50 cm from the patient surface to avoid the skin - sparing effect . Total prescribed dose of 12 gy (150 cgy per fraction) was delivered to the patients over eight fractions of treatment in 4 days . Each day, two fractions of treatment were given with a gap of minimum 6 h. treatment was delivered at a dose rate of 6 cgy / min at the treatment distance by setting 100 monitor units (mu)/min in linac . Number of mus required to deliver the per field prescription dose of 75 cgy was in the range of 13001400 mus depending on the average thickness of the patient . During patient preparation, anterior posterior (a - p) thickness was measured at forehead, neck, chest, abdomen, pelvis, thigh, knee, and ankle to calculate the average thickness of the patient . Test dose of 20 cgy (10 cgy from anterior field and 10 cgy from posterior field) was delivered to the patients prior to treatment for dosimetric verification . Tld chips are the standard detector used for patient in vivo dosimetry in our tbi treatments . In vivo dosimetry was carried out by pasting tld chips at multiple sites throughout the patient body to measure the dose received . After ensuring acceptable dose results from the test dose measurements, patients were taken up for further treatment . Radiation output and depth dose profiles of the linac measured at the tbi setup were used for the mu calculation . Percentage depth dose (pdd) values measured for standard 100 cm ssd and converted for 400 cm ssd using the mayneord factor differ from the actual pdd for tbi setup, as the difference in ssd is very large . Hence, it is recommended that a direct measurement of the pdd and output should be obtained at the tbi setup . Rfa-300 radiation field analyzer (scanditronix wellhofer, uppsala, sweden) having mylar window on one wall was used to measure the pdd at the tbi setup . High - doped p - type silicon diodes (hi - p si) were used as the detectors . The reference detector was also kept beyond the beam spoiler (close to the phantom) in the same spectrum of beam energy . Accuracy of pdd data measured using diode was verified using gafchromic films at multiple depths (absolute measurement). Pdd values generated using mayneord factor for 400 cm ssd at every 5 mm were compared with the pdd values measured for tbi setup [figure 1]. Also, it was observed that for the pdd measured at tbi, the depth of dose maximum (dmax) had shifted to 4 mm depth compared to 15 mm depth for the pdd generated using mayneord factor . Based on these observations, the bolus material to keep the tld during in vivo dosimetry was made of 5 mm thickness to achieve buildup . Comparison of pdd curve obtained at tbi setup and pdd curve obtained at 100 cm ssd and converted using mayneord factor output measurement of the linac at the tbi distance was measured using solid water slabs (density: 1.03 g / cc) of dimensions 30 cm 30 cm . Multiple solid water slabs were used to make a phantom of 20 cm thickness . A 0.6 cc cylindrical ionization chamber (ptw, freiburg, germany) with unidos e electrometer was used for measurements . Care was taken to reduce the direct exposure to dosimetry cable to minimize the leakage . The ionization chamber was kept at a depth of 10 cm and irradiated with 500 mu . The meter reading obtained was used to calculate the radiation output using the pdd value obtained at 10 cm depth for tbi setup . Iaea trs-398 formalism for output measurement of high - energy photon beams was used to calculate the output taking into account the ion recombination, polarity correction, and temperature and pressure correction . Two sets of lif tld-100 chips (harshaw, solon, oh, usa) of dimension 3.2 mm 3.2 mm 1 mm were used in this study . Tld chips were annealed at 400c for 1 h using fisher scientific thermal furnace, followed by gradual cooling at room temperature . A semi - automatic tld reader (harshaw 3500) was used to read the response from the tlds . Prior to start using the tld chips for dosimetry, both sets of tld chips were calibrated in order to establish dose as a function of tld reading . All the tlds were then annealed and irradiated with 6 mv photon at 5 cm depth in solid water phantom (density: 1.03 g / cc) with a 10 cm 10 cm jaw opening in the linac at 100 cm ssd to a dose of 200 cgy . During tld calibration and tbi in vivo dosimetry tld chips showing response within 1% of the mean response of the entire set were chosen as golden chips . After identifying the golden chips, tld chips were again annealed and irradiated in the standard setup to a known dose . The reader response to these golden chips was used as the calibration factor for the tld reader . The remaining tld chips other than the golden chips, known as field dosimeters, were read to generate their unique calibration factors . These field dosimeters were then used as the detectors in tbi in vivo dosimetry . A glow curve image obtained from the tld reader during tld calibration dosimetry results of 20 tbi patients (aged between 3 years and 28 years) treated between march 2008 and december 2011 were analyzed in this study . The intention of the in vivo dosimetry check was to ensure that the prescription dose was delivered accurately and uniformly throughout the patient's body . Hence, before using tld for every tbi patient, both sets of tlds were exposed to a known dose (200 cgy) at the standard setup used for tld calibration . Subsequently, only those tlds whose response fell within 3% of the given dose during this sensitivity check were used for the in vivo dosimetry . Tlds were recalibrated if the whole set of tlds showed more than 3% difference between the given dose and the measured dose during this sensitivity check . The measured dose values by each tld set during the multiple sensitivity checks performed were averaged out and are shown in figure 3 . Average of the measured doses by each tld during the sensitivity tests tlds along with wax bolus over each tld were kept at forehead, mediastinum, chest, abdomen, pelvis, thigh, and calf during in vivo dosimetry . Thickness of the bolus was maintained as 5 mm to achieve buildup region . From the results, minimum, maximum, and average measured dose values at each anatomical site prescription dose was divided into different ranges (<95%, 95105%, and> 105%), and an analysis was made for all sites on how many number of times the measured dose falls in each dose range . Mean dose to the whole body with standard deviation was calculated for each fraction of the treatment to check the homogeneity in the dose delivery . Table 1 shows the minimum, maximum, and average measured dose at each site . Due to the large variation in body thickness and internal tissue heterogeneities, the minimum dose and maximum dose measured at each site had a significant variation (> 10%) from the prescription dose . However, an analysis on the average measured dose from each site among the whole data set reveals that the average dose to each site falls between 96% and 102% of the prescription dose . Minimum, maximum, and average measured dose at each site results obtained from the tld kept on the patient's forehead show an average measured dose value of 97.33% . The average measured dose at mediastinum was 96%, with 95.65% at the anterior mediastinum and 96.4% at the posterior upper chest . Dose measured at the right chest was found to be about 1% higher than the dose measured at the left chest . Dose recorded from abdomen and pelvis followed similar patterns . The average measured dose levels at umbilicus, posterior pelvis, and abdomen were 96.07%, 96.75%, and 96.99%, respectively . Eight tlds were used for the legs, four on the thigh and four on the calf . The average measured dose values to thigh and calf were 97% and 101%, respectively . It is observed that the minimum and maximum dose measured at each site varied significantly from the average dose for that site (e.g., the difference between average dose and minimum dose for left posterior calf was more than 16%). Hence, to understand how frequently the dose measured at each site is deviating from the average dose, results for each site are distributed into different dose ranges [table 2]. An analysis of the same would give an idea on what dose shall be expected from each site . In most of the instances (more than 80% of the time), the dose measured at mediastinum, abdomen, forehead, and pelvis falls between 95% and 105% of the prescription dose . This pattern was followed by thigh (68.8%), chest (66.3%), and calf (65.9%) for recording 95105% of the prescription dose . Frequency of occurrence of measured dose within a dose range (in%) though the minimum dose for each site from the whole data is below 95% of the prescribed dose, this occurred on a few occasions only (6.519.2% of the measurement times for different sites). The prescription depth is significantly different from the midline depth for sites like pelvis and calf, as the dose is prescribed to the midline depth of the patient's average body thickness . When the thickness of the pelvis is larger than the average patient thickness, a lesser dose is expected to be measured from the tlds kept on pelvis . Thus, tlds kept in pelvic region recorded a dose less than 95% of the prescription dose on more occasions compared to tlds from other sites . In spite of the proper instructions given to patients, on few occasions, there is a possibility of patient movement during the treatment due to the long duration of the treatment time . This movement of the patient can also result in a significant variation in the measured dose . In calf, chest, and thigh, this resulted in the tlds kept at calf to record a dose as high as 110% of the prescribed dose . Calf used to be the region to receive the maximum dose among all the sites in a single fraction . The same reason of lesser thickness is applicable for the tld kept on thigh also (for more than 15.8% of measurement times, the measured dose at thigh was greater than 105% of the prescription dose). Another site in which the dose recorded was greater than 105% of the prescription dose is chest . In other sites, the measured dose was higher than the prescription dose on fewer instances and it was always less than 5% . There are few inherent uncertainties in the tld dosimetry system in its various stages like annealing, calibration procedure, and tld readout . Have reported that 2% uncertainty in the measured dose and 5% action criterion for tld dosimetry are reasonable while using tld for absorbed dose measurements . Measurements at many sites, particularly chest and pelvis, correlated well with the study results of lancaster et al . Dose to chest in our measurements was 99.4% compared to 99.3% in their measurements using semiconductor diodes . Dose levels measured on pelvis, abdomen, and head showed the lowest agreement with the study by lancaster et al . Similarly, these are the sites in which the dose measured was less than 95% of the prescribed dose on more occasions in our measurements . Homogeneous dose delivery to the whole body is a requirement in tbi . To check the uniformity in dose delivery, mean dose of the doses measured from all sites was calculated with standard deviation for each patient . The standard deviation is given in cgy, and it indicates the variation to which the dose is delivered uniformly throughout the body . The mean dose to whole body (135.5151.7 cgy) was close to the prescription dose for most patients and was within 10% of the prescription dose for all patients in our tbi in vivo dosimetry . Figure 4 shows the mean dose with standard deviation values calculated for all patients . Though dose homogeneity along the body axis within 10% is acceptable for most of the tbi protocols, this was significantly worse compared to the in vivo dosimetry results reported by palkoskov et al . The main factor worsening the dose homogeneity in the technique used in our tbi protocol was the dose to extremities and chest . The tbi treatment protocol used in this study is simple and easy to implement . In any single fraction of tbi with the treatment technique used in this study, a dose within 5% of the prescription dose is expected to be measured from all the sites except calf and chest when tld is used for the in vivo dosimetry . The practice of using lif tld-100 for in vivo dosimetry is a good option for tbi dosimetry . The same could be used in similar quality assurance checks for other techniques in radiotherapy, if the tlds are properly calibrated and checked for the particular technique . With our in - house tbi protocol of ap a better homogeneity in dose delivery is possible by adding tissue compensators for extremities and chest . Also, accurate and more consistent results could be expected if a detector which has less uncertainty in measuring the delivered dose is used . With this observation, we have started exploring the use of gafchromic ebt2 film by using the film along with tld in our tbi dosimetry, and the results can be analyzed to standardize the technique in future.
The classification and the terminologies used for various vascular lesions have been very confusing despite the fact that a biological classification was first published in 1982 by mulliken and glowacki . This classification was later adopted by the international society for the study of vascular anomalies (issva) in their first workshop held in rome during june 1996 . This continuing workshop now takes place every 2 years in various countries around the world . The issva is an organization comprising of specialists in various disciplines interested in vascular anomalies and was founded in 1992 in budapest with the aim of achieving consensus among health care professionals on the terminology, to further the knowledge of pathogenesis, diagnosis and treatment of these vascular lesions . An analysis of various scientific articles and latest edition of text books showed that significant confusion still prevails due to the indiscriminate, inappropriate and interchangeable use of various terms . Historically benign vascular tumors were classified: (1) according to the type of fluid they contained as hemangioma (blood - containing lesion) and lymphangioma (lymph - containing lesion) and (2) according to the size of the vascular channels as capillary (small diameter vascular channels) and cavernous (large diameter vascular channels). Mulliken and glowacki described a biological classification based on endothelial cell characteristics, physical findings and natural history, that differentiates vascular lesions with endothelial cell proliferation (example hemangioma) from lesions with structural anomalies (vascular malformations). The issva modified it in their continuing workshops, differentiating vascular tumors from vascular malformations based on their clinical appearance, radiological features, pathological features and biological behaviour [table 1]. Historically benign vascular tumors were classified: (1) according to the type of fluid they contained as hemangioma (blood - containing lesion) and lymphangioma (lymph - containing lesion) and (2) according to the size of the vascular channels as capillary (small diameter vascular channels) and cavernous (large diameter vascular channels). Mulliken and glowacki described a biological classification based on endothelial cell characteristics, physical findings and natural history, that differentiates vascular lesions with endothelial cell proliferation (example hemangioma) from lesions with structural anomalies (vascular malformations). The issva modified it in their continuing workshops, differentiating vascular tumors from vascular malformations based on their clinical appearance, radiological features, pathological features and biological behaviour [table 1]. Hemangiomas grow by endothelial cell hyperplasia and should be differentiated from vascular malformations, which are not true neoplasms but are localized defects of vascular morphogenesis caused by dysfunction in embryogenesis and vasculogenesis [table 2]. The greek suffix means cellular proliferation of a tumor and thus the term hemangioma is erroneous when used for malformations . Hemangiomas are the most common benign soft tissue tumor of infancy and childhood, occurring in 12% of all infants and are found in greater frequency in girls, whites, premature infants, twins and are usually born to mothers of higher maternal age . They occur most frequently in head and neck region (60%), followed by the trunk (25%) and the extremities (15%), which are grouped into infantile hemangiomas (ihs) and congenital hemangiomas (chs). Differences between infantile hemangioma and vascular malformations ihs (outdated term juvenile hemangioma) arises during the first 8 weeks of life as an area of discoloration or telangiectasia . The lesion exhibits a rapid proliferative phase during early childhood for 6 - 12 months and grows into a raised rubbery bright - red tumor (resembling a strawberry, hence outdated term strawberry hemangioma). This is followed by gradual involution and a spontaneous regression by the age of 5 - 9 years . 50% of all hemangiomas will completely involute by the age of 5 years and 90% by the age of 9 years . 40% of involuted lesions may either show scaring, wrinkling, telangiectasia, or loose fibro - fatty tissue . Ihs can be grouped into focal, segmental and indeterminate, or depending on the depth of the lesion from the skin surface as superficial, deep and mixed . Focal ihs are the most common variant, appearing as localized raised tumor - like lesion that tends to occur at the area of embryological fusion . Segmental ihs are flat plaque - like larger lesions that show a geographic segmental distribution and indeterminate ihs shows characteristics of both focal and segmental ihs . Color varies with the depth of the lesion; they can be bright red (superficial), purple, blue, or normal skin colour (deep). Chs are clinically present as fully developed lesions at birth and either rapidly involutes during the first year of life or may never show involution . These lesions do not exhibit a proliferative phase and do not grow after birth . Rapidly involuting congenital hemangiomas (rich) are present at birth, either as red - purple color plaques with coarse telangiectasia, or as flat violaceous lesions, or as a raised greyish tumor surrounded by a pale halo with multiple tiny telangiectasia . Rich undergo a rapid regression phase and completely disappear by 12 - 18 months of age . Non involuting congenital hemangiomas (nich) are also present at birth, appearing as pink or purple colored plaque - like lesions with prominent overlying coarse telangiectasia and peripheral blanching . Nich do not show a regression phase and grows proportionately with the growth of the child . Differences between congenital and infantile hemangioma apart from hemangiomas of soft tissue, scientific literatures have reported central hemangiomas (hemangioma of bone) and intramuscular hemangiomas . Many researchers in addition to the world health organization (who) believe that most if not all such proposed lesions are vascular malformations rather than true neoplasms . Therapeutic guidelines, management and follow - up of hemangiomas and vascular malformations differ and are beyond the scope of this article . A good classification is important for categorising information, recording data, proper communication, guiding treatment plans, obtaining prognostic information and should be easy to understand and applied by the clinicians . It is our duty to be consistent in our terminology and classification of vascular lesions in all our scientific writings and presentations in order to communicate effectively, understand its pathophysiology, promote research and develop newer therapeutics . Terms to be avoided when describing these lesions include angioma, birthmarks, capillary hemangioma, cavernous hemangioma, juvenile hemangioma, strawberry hemangioma and inappropriate interchangeable use of the terms hemangioma and vascular malformation.
Gunshot wounds to the spine account for approximately 13 to 17% of all spinal cord injuries each year5). Spinal cord injury after gunshot usually results in complete injury, such as paraplegia, which is highly associated with thermal tissue injury34). However, neurological deficits can be variable, ranging from radiculopathy to spinal cord injury . Although neurological deficits might be absent initially, most patients eventually develop deficits that require surgical intervention6). We describe a rare case of delayed onset of neurological symptoms after a bullet injury to the flank; the bullet migrated and became lodged in the spinal canal . We also review the relevant literature and compare the current case with other clinical case reports . A 30-year - old man presented to our outpatient clinic with radiating pain both legs . The pain in both legs was aggravated with walking and relieved with rest, symptoms typical of neurogenic claudication . The patient reported that he had been shot in the left lateral side of the back on 1 year ago . We determined on physical examination that the wound on the left flank had already healed (fig . 1). At the time that he was shot, the patient had no other complaints, except for pain at the entrance wound . At that time of gun shut, he had to have absolute bed rest and conservative treatment because of a liver injury caused by the gunshot . After several months of treatment, his liver was healed enough to ambulate and walk; however, he needed a cane to walk . After he started walking, he began to experience continuous radiating pain on both lower extremities . On neurologic examination, dorsiflexion of the ankle and big toe on the right leg revealed mild motor weakness (grade iv). The patient also complained of numbness on the right lower limb; however, patellar and achilles reflexes were normal . On admission, other laboratory findings were normal . A mild degree of voiding difficulty was detected, although that symptom did not bother the patient . Plain radiographs revealed a round, radiopaque mass in the spinal canal at the l3 - 4 level (fig . Non - contrast reconstructed ct images of the lumbar spine were taken after admission, which revealed that the bullet - shaped mass was within the spinal canal, pointing cephalic at the l3 - 4 interspace (fig . The bullet within the spinal canal was located in the intradural space, compressing the nerve root (fig . Because of his claudication and pain, we decided to surgically remove the bullet from the spinal canal . With the patient prone on a wilson frame, fluoroscopic guidance was used to plan an incision over the l3 - 4 spinous process . The l3 lamina was carefully and completely removed with a drill, and a dural incision was made . Both intraoperative and postoperative motor - evoked potential and somatosensory - evoked potential were normal . The patient ambulated the day after surgery and reported that the radicular pain and claudicating pain were greatly improved . He was discharged 1 week after surgery . On his follow - up visit at 4 weeks, the pain had nearly resolved, and oral medications, including pain killers, were completely stopped . He showed definitive improvement in motor power of the right leg; however, independent ambulation without a cane was not achieved . The annual incidence of spinal cord injuries is 10.4 and 83 per million people, and the most common cause of spinal cord injuries is motor vehicle accident910). Spinal cord injuries due to gunshot wounds are very rare, and the absence of acute neurological symptoms after gunshot - related spinal injury is even rarer . Our case report is particularly interesting because intradural localization and our patient developed radiating pain in both lower extremities 1 year after the initial injury . In additions; to our knowledge, only a few cases with similar characteristics have been reported27). Most bullet injuries are accompanied by acute neurologic symptoms, and if initially the patient might be neurologically intact fortunately, neurological symptoms gradually appear over time . There are two possible explanations for the delayed appearance of neurological symptoms in the literatures: first, compression of the spinal cord or the thecal sac and impingement of the nerve root as a result of migration of the bullet and second, reactionary epidural fibrosis . According to a paper by ajmal et al.1), there have been only 7 reported cases of delayed neurological symptoms caused by epidural fibrosis after gunshot injury . First, our patient developed radiating pain in both lower extremities 1 year after the initial injury, and the cause was thought to be epidural fibrosis due to the injury itself . Second, it is unique that our case is intradural bullet injury . In the surgical field, there was a significant inflammatory response in the area where the bullet was lodged . However, since serial images were unavailable, we could not compare images taken when the patient incurred the injury with the images taken in our clinic; as a result, we were unable to completely exclude true bullet migration or potential stenosis because of degenerative changes . We presumed that bullet might enter the body of left flank, which would be the level of thoraco - lumbar junction (fig . 1), because of the entrance skin wound . After penetrating skin on left flank of thoraco - lumbar junction, bullet might penetrate the vertebra and dura, which was finally found with adhesion on l3 - 4 level . We assumed that bullet might be located at intradural thoraco - lumbar junction at first injury, which migrated on l3 - 4 level after ambulation . After migration on l3 - 4 level, bullet was stuck to l3 - 4 level, because of its size and diameter . And epidural fibrosis and intra dural adhesion might follow . When determining the surgical treatment for patients with bullet injury, surgeons should assess neurological damage and abnormalities, instability, the position of bony fragments, age, and systemic complaints . Ajmal et al.1) opposed surgical removal of intraspinal bullets because the injury is caused neurological deficit in the end, but surgical treatment should be considered when patients have instability and neurological damage or abnormalities, whether delayed or immediate . In an acute setting, the indications for exploration include, but are not limited to, cerebrospinal fluid leak, infection, and partial or progressive deficits . Risk of bullet migration and development of fibrosis and stenosis over time should be considered in other patients . These patients should therefore be informed about late complications if a decision is made to manage them conservatively . Regular follow - ups with radiographs may also be required to monitor bullet migration18). In contrast, ben - galim and reitman2) recommend early surgical intervention even if the patient has no neurological symptoms initially because of the risk that neurological deficits will develop as a result of intrathecal bullet migration and inflammatory reaction . In our case, after healing of the internal organs, the patient began walking with a cane, after walking he discovered that he had continuous radiating pain in both lower extremities . We presumed that a claudication patient needed bed rest for healing of internal organ injury, which masked the walking symptom of claudication and radicular pain . We decided on surgical treatment because the patient had neurologic abnormalities and neurological symptom aggravation . After surgery, he showed definite improvement of motor power in the right leg; however, independent ambulation without a cane was not achieved . Firstly we found the inflammatory changes in surgical field, however we missed taking pictures ., we could not find and get the initial radiology of initial gun shot, which would be the good evidence of the pathophysiology in this case, whether symptom develop form migration of bullet or inflammation, however we presumed that both would be related with symptom . We documented an extremely rare case of intradural location of a bullet, which was treated surgically with a satisfactory outcome . We need to be cautious, if you see the non - symptomatic gunshot patients without penetration of bullet, which means absence of outlet of bullet . From current case and reviews of literatures, we need to think about possibility of intradural migration of bullet or chronic inflammation and adhesion to surrounding tissue.
A coracoid fracture is a rare orthopaedic condition as it accounts for only 2 10% of all scapular fractures and <0.1% of all fractures . In even rarer cases coracoid fractures we present an unusually rare case of a simultaneous fracture of the coracoid process and the lateral end of clavicle . The patient was treated with shoulder arm sling for 6 weeks followed by physiotherapy . At 6 months follow up the patient was pain free with full range of motion at right shoulder . Concomitant fractures of lateral end clavicle and coracoid process are rare and may not be visible of plain radiographs . Ct scan may be indicated if the clinical examination suspects additional injuries of shoulder girdle . Coracoid fractures alone are themselves a rare occurrence amounting to approximately <0.1% of all fractures and even only 5% of all fractures of the shoulder [1 - 3]. No reliable figures currently exist to determine the prevalence of a simultaneous distal clavicle and coracoid fracture . A 60 year old gardener presented with a history of injury to her shoulder as her arm was pulled by a lawn mower . The patient did not have any previous injury or degenerative condition to this shoulder prior to this accident . Physical examination showed swelling and bruising around shoulder, tenderness and restricted movements of the right shoulder . Ct and plain radiographs of the shoulder displayed a displaced coracoid fracture and non - displaced fracture of the distal end of clavicle at the time of injury (fig . The patient was placed in a shoulder arm sling for six weeks and was then referred to physiotherapy . Some doubtful angulation of the coracoid process is visible but fracture is not appreciated clearly . 1b- ct scan conforms the fracture of lateral end of clavicle and also displace coracoid fracture . At three months the patient was pain free and back to working as a gardener with a free range of movement in the right shoulder . 3 months follow up radiographs showing no further displacement of the fracture with early consolidation at the fracture site . When fractures of coracoid and distal clavicle co - exist, misdiagnosis is common because both the fractures may not be seen on standard projections . The stryker notch view is recommended for detection of these fractures . Furthermore ct imaging is advised if radiographs initially fail to show any bony injuries of the shoulder, but clinical examination suggests otherwise . The mechanism of injury is probably the result of a resisted flexion of the arm and elbow which led to a forceful pull of the muscles, pectoralis minor and the coracobrachialis, that insert into the coracoid . This has previously been described in two other case studies concomitant coracoid fractures and acromioclavicular dislocation . The patient suffered an ipsilateral avulsion of the coracoid which would be considered as a type ii according to ogawa et al and a distal clavicle fracture which would be considered as a type i according to the neer s classification . It is to this day still controversial how to treat coracoid or distal clavicle fractures individually, let alone if they are present concomitantly . Details and consequent treatment methods of coracoid fractures have also yet to be established in literature . Ogawa et al have recommended surgical treatment of coracoid fractures combined with other shoulder injuries, but have otherwise shown similar outcomes between operative and non - operative treatment of coracoid fractures alone . They did however recommend non - operative management of type ii fractures as these do not disturb the scapuloclavicular connection . There does not seem to be a general consensus on the treatment of distal clavicle fractures too . Robinson et al have recommended non - operative management of distal clavicle fractures in the middle aged or elderly patients . Out of the 101 patients treated non - operatively with distal clavicle fractures, only in 14% of cases rokito et al compared nonoperative and operative treatment of a total of 30 type ii distal clavicle fractures retrospectively and did not reveal any significant difference in terms of ucla, constant and asesscore . On the other hand haidar et al and meda etal advocate the use of clavicular hook plates . These studies however did not compare the operative treatment to non- operatively treated control groups [10, 11]. The option of treating a symptomatic nonunion of the distal clavicle was described by kang et al who achieved 100% bone union in 10 patients following orif with an oblique locking t - plate and autogenous bonegraft . Lasda et al described successful treatment of a concomitant acromioclavicular dislocation and coracoid fracture conservatively . Eleven years later carr et al further described two cases of concomitant coracoid fracture and acromioclavicular dislocation which were treated conservatively with good results . Recently duan et al published a case report of operative treatment of an acromioclavicular dislocation in combination with a coracoid fracture which was treated with a hook plate . They described the problem of only partial bone union after 5 months and the additional plate removal that needed to be done 12 months after the initial operation . Only one case report by ruchelsman however has described the operative treatment of a concomitant lateral clavicle and coracoid fracture . In his case he then proceeded with a open distal clavicle excision and reduction of the coracoclavicular interval with screw fixation achieving an excellent clinical outcome . They commented that high index of suspicion is needed to avoid missing such combination injuries at shoulder girdle and additional radiological investigations should be done if pain at shoulder girdle is persistent . In view of the sparse and controversial literature available, we believe that the patient should be appropriately informed of both operative and nonoperative options treatment options . In our case the patient preferred nonoperative management which resulted in an excellent clinical result . Concomitant fractures of lateral end clavicle and coracoid process are rare and may not be visible of plain radiographs . Ct scan may be indicated if the clinical examination suspects additional injuries of shoulder girdle . Even in cases with displacement a conservative management will result in a good clinical outcome as seen in our case.
A spinal cord injury that affects mainly the final common motor pathway and the primary sensory trajectories occurs as the spinal nerve roots are torn or avulsed from their medullary attachments . In humans this occurs most frequently in traction injuries to the brachial plexus but also in trauma to the lumbosacral plexus and cauda equina as well as conus medullaris . Root avulsion interrupts the local transverse segmental spinal cord motor and sensory nerve fibers and is a longitudinal spinal cord injury . This injury has a serious effect of the pertinent spinal cord segment with the breakdown of connections and networks and the development of a spinal cord scar . Disintegration of the shorter central part of the sensory axon and most of the motoneuron axon with a dying back into the spinal cord follows . Within a few weeks after such a trauma there will be an increasing death of neurons in the spinal cord, affecting motoneurons, and autonomic neurons in the ventral horn as well as sensory neurons in the dorsal horn . There is, however, very little if any disintegration of the primary sensory neurons in the ganglion in the dorsal root (carlstedt, 2007). The functional consequence of such injury is lower motoneuron syndromes, associated with autonomic paralysis including dysfunctional internal organs, limb muscle atrophy, sensory impairment, and chronic pain . These injuries most often occur as a result of road traffic accidents or violent acts . Nerve root injury has been associated with an overall poor clinical outcome as a successful surgical repair would require axonal regrowth within the central nervous system as spinal cord regeneration . Today, therefore, most patients with this type of injury are not treated at all or receive non - curative palliative surgery, which do not give the same level of recovery and pain alleviation . Experimentally, it was obvious that after dorsal root avulsion and repair sensory axons when encounter the astrocyte dominated cns part of the root are immobilized and seem to be stabilized, rather than repelled it seems as if they stop because of synapse formation which might be more instructive in the inhibition than inhibitory molecules or lack of intrinsic regenerative capacity . Electron microscopical analysis showed that at least some of these axons form nerve terminals exhibiting active zones which contain transmitter substance . This is another way of inhibition than what currently is the concept (he and koprivica, 2006). There are a number of reports of induced regeneration of dorsal roots by transplantation of glia cells (kliot et al ., 1990), conditioning lesions (chong et al ., 1999), or application of growth factors (ramer et al ., 2002; wang et al a type of stem cells, the olfactory ensheathing cells (oec) when applied to dorsal roots after injury aggregate in the transitional region without entering into the spinal cord and seem to form a scaffold for the regrowing sensory fibers which can cross into the spinal cord . This technique is now just about to be applied clinically for restoration of sensory function after root injury (li et al ., 2004). The intracellular neurophysiological and staining technique that unequivocally demonstrated that defined injured spinal cord motoneurons after avulsion and replantation of detached ventral roots could regrow new central nervous axons is the golden standard by which any claim of cns regeneration should be assessed . In those experiments restoration of muscle function by the injured motoneurons the recovery of autonomic visceral function was demonstrated from the application of this surgical method in conus medullaris and cauda equina injuries as re - innervation of the lower urinary tract occurred (hoang et al ., the biological background to such exceptions to the rule of unsuccessful regeneration in the cns remains partly elusive . A human surgical strategy for the repair of avulsed ventral roots and this line of work has been successfully continued in clinical studies and a real translation of laboratory experiments and results to clinical cases was commenced about 20 years ago (carlstedt et al ., 1995; carlstedt, 2007). By the replantation of avulsed ventral roots or reconnection by means of nerve grafts between the pertinent spinal cord segments and the avulsed ventral roots there was recovery of useful function mainly to proximal limb muscles after brachial as well as lumbosacral plexus injuries . The best results have been noted in a few preadolescent patients where useful hand function was restored from replantation of lower spinal roots to the brachial plexus . Neuropathic pain commonly follows avulsion injuries to the brachial plexus as well as cauda equina injuries . Interestingly, there is a significant correlation between pain intensity and the number of avulsed roots . The mechanism behind nerve root avulsion induced pain is not understood but there is a conspicuous alleviation of this pain with recovery of muscle but not sensory function (berman et al ., 1998; htut et al ., 2006). Thus in patients with a complete brachial plexus avulsion injury where replantation has been performed there is in conjunction with restored muscle activity diminished pain (havton and carlstedt, 2009). In patients with re - established muscle function throughout the extremity there is a total loss of pain (carlstedt et al ., 2004). Basic science correlates to this clinical observation has recently been done as ventral root avulsion is capable of eliciting damage that propagates from the ventral to the dorsal horn and can cause degeneration in primary afferent fibers (bigbee et al . Interestingly, ventral root implantation protects against afferent degeneration, and reduces the glia response as well as promotes motoneuron survival (bergerot et al ., 2004; is poor as restoration of primary sensory neuron contacts in the dorsal horn has clinically not been possible . Intriguing is, however, that even without sensory feedback such as proprioception from muscles there was recovery of useful function also in the hand when exclusively motor trajectories were restored from spinal cord implantation . Functional mri of cortical activities in such a case has been performed (carlstedt et al ., 2009). Not surprisingly it was shown that when the affected hand was used there was bilateral cortical activities which did not happen in the normal situation . This is related to cortical plasticity as there is a bilateral cortical representation for each side but normally the ipsilateral cortex is inhibited through transcallosal connections which in circumstances such as multiple sclerosis and stroke are disinhibited . There was also and more remarkable activity in the sensory cortex when the deafferentated hand was active . This finding demonstrates in fact that there is a cortical sensory program that acts independently of afferents and is serving motor commands . This is of conceptual importance and means that proprioception is not obligatory for muscle function and movements . Most likely such cortical sensory programs are also active in the normal individual in many movements . If for every movement proprioceptic input would be necessary according to the original concept there would be a substantial time delay and many mistakes done . It is therefore likely that as also has been described in experimental situations there is a direct sensory motor cortex interaction depending on an established sensory program in the cortex for rapid motor performances which of course in the normal situation is tuned properly by proprioceptic input particularly in dexterous and fine manipulative tasks . Thus, surgical replantation of avulsed cervical and lumbosacral ventral spinal roots can promote spinal cord or central nervous regeneration of autonomic and somatic motoneurons with functional re - innervation of peripheral targets such as muscles and pelvic organs . Unexpectedly, and as it seems fringe benefits from such surgery are pain alleviation as well as central cortical proprioceptive modulation of restored muscle activity . Both central nervous system regeneration and muscle function without proprioceptive function are of conceptual importance, but there is room for improvements to augment functional recovery particularly regarding restoration of sensory function but also motor activity . The surgery to replant avulsed ventral roots to the spinal cord has a documented effect on motoneuron survival (bergerot et al ., 2004; bigbee et al ., 2008; eggers et al ., 2008). This effect is, however, transient and in long time follow up there is no real difference in motoneuron population after avulsion only and avulsion with root replantation (bergerot et al . The amount of motoneurons surviving after avulsion injury was found to be lower in situations where also the dorsal root had been avulsed (gu et al ., 2004) or when neurons expressing chat immunohistochemistry were assessed (hoang et al ., 2006 this discrepancy could depend on different surgical techniques but most likely how the motoneuron population was estimated as quantification varies due to the staining method, with immunohistochemistry revealing greater loss than histological dye . Brain derived neurotrophic factor (bdnf) but in particular glia derived neurotrophic factor (gdnf) has experimentally proved to be effective in motoneuron maintenance . In combinatorial experiments when such substances were given to animals after root avulsion and replantation there was a good persisting effect with improved functional outcome (bergerot et al ., 2004). Several already approved pharmacological products have been tested experimentally in root avulsion with longitudinal spinal cord injury . One of these, riluzole, which currently is used for amyelotrophic lateral sclerosis, has shown a good neuroprotective effect after root avulsion (bergerot et al ., 2004). Interestingly, a combination of riluzole and gdnf applied after ventral root avulsion and replantation showed a good, robust regeneration . Using two different locomotor tests it was found that significant functional recovery approaching normal values occurred only after re - implantation of avulsed ventral roots together with gdnf and riluzole treatment . These results show that functional recovery is correlated to enhanced dendritic complexity and increased survival of motoneurons . Riluzole promotes dendritic outgrowth and neurotrophins such as gdnf promote motoneuron survival and axonal regeneration and both are required for functional recovery possibly by increasing neurotransmitter level or synthesis in motoneurons . Combinations of substances which provoke such neuronal reactions are needed to achieve a good outcome from repair of root avulsion injury . Thus, combination treatment may offer a new therapeutic strategy for treating patients with avulsion injury . For instance a gene therapy approach using adeno - associated viral (aav) vectors for long time delivery was indeed successful in preventing motoneuron death due to the trophic effect of the secreted neurotrophic factors, but the regenerated axons were locked up in the vicinity of the delivery site of the viral vectors and there was no distal growth and consequently no functional restoration . This may be solved by creating a longitudinal gradient of the neurotrophic factors by using a controllable or transient gene product . However, even if neurons are protected functional recovery is presently limited to proximal regions . This is due to a restricted amount of regrowing axons as well as the slow rate of regeneration leaving distant targets denervated for a long time . Chronically denervated schwann cells cannot maintain correct neurotrophic support and distal muscles become atrophied and fibrosed with eventually very little intact muscle to innervate (suliman and gordon, 2000). In preliminary studies using oec applied to the site of ventral root implantation there was a four - fold increase in regenerating axons (li et al ., 2007). It is of course not yet clear whether the increased number of motor fibers induced by presence of transplanted oecs in the re - implanted ventral root would per se overcome the lure of simply increasing the innervation of the nearer proximal muscles (eggers et al ., 2010). Strategies should however not only be directed to problems of neuroprotection and augmenting number of regenerating axons . If the pace of regrow cannot be increased means to save denervated muscles should be implemented . There are now preliminary data that already existing drugs could have such effects (shortland, personal communication) in spite of the reserve cortical sensory capacity which helps in more simple movements as well as in alleviation of pain through corrections of motor trajectories, it is necessary to try to re - establish the afferent loop of the damaged spinal cord connections . Most promising in experimental work has been the application of oec, a type of stem cells, which has promoted re - entry of primary sensory axons into the dorsal horn of the spinal cord (li et al ., an interposition of oec between the schwann cells and the astrocytes induced bridging channels from the pns to the cns parts of the root that allowed dorsal root nerve fibers to enter the spinal cord (li et al . This arrangement appears similar to the naive arrangement at the tr of schwann cell columns surrounding individual nerve fibers in the channels of the astrocytic fringe . Regenerated nerve fibers could be demonstrated within the dorsal horn as well as within the white matter of the dorsal columns . Functional restoration has also been reported with this technique as progressive improvements in paw grasping a frame bar during climbing (ibrahim et al ., 2009). An alternative method to re - establish sensory connectivity would simply be to discard the primary sensory neuron, i.e., to excise the dorsal root ganglion, and replant the dorsal root into the dorsal horn to allow secondary sensory neurons to extend into the periphery . This was previously demonstrated in animal experiments (carlstedt, 1985b) and has according to preliminary data with some success recently been applied clinically . Interest should also be directed to the effect on second and third order sensory neurons from the impact of sensory root avulsion . It has been documented that avulsion rather than rhizotomy leads to about 30% death of dorsal horn cells (chew et al ., 2008). There is today no information about a transynaptic effect of such trauma which of course is crucial once regeneration of primary sensory neurons into the dorsal horn is established . Maintenance of secondary sensory neurons is obviously also of importance with regards to the classical excruciating avulsion pain which today is poorly understood . Of interest in this context is also the role played by increase in glia activity in the dorsal horn in particular by microglia . For instance with microarray techniques the complicated biological events resulting from gene activity after injury and repair can be assed and valuable insight and cues how to advance would be gained . With regards to curtailed or restricted axonal growth within the spinal cord some of those are small lipophilic molecules acting as receptor agonists, which potentially can reach injured neurons without difficulties in administration or dosage . For instance, functional regeneration of sensory axons into the spinal cord was achieved after stimulation of the retinoic acid receptor by such compounds (wong et al ., 2006). A caudal spinal cord injury at conus level has many similarities with the root avulsion as mainly the lower motoneurons and cns parts of the peripheral sensory neurons together with nervous control over bladder and bowel function is affected . This type of injury will in the future be considered for similar type of surgical strategy in man as for the spinal cord injured patients control over pelvic organs has the highest priority . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Hepatolithiasis, that is, the formation of stones in the intrahepatic biliary tree, is most prevalent in east asia, including china, japan, and korea (1). The goals of primary treatment are to resolve ongoing infections and to prevent recurrent cholangitis, subsequent hepatic fibrosis, and progression to cholangiocarcinoma (2). However, complications associated with hepatolithiasis, such as recurrent cholangitis, hepatic cirrhosis, and cholangiocarcinoma, frequently occur even after effective treatment . Long - term complications are usually considered to result from inadequate stone retrieval, concurrent biliary stricture, or long standing cholangitis (3, 4). However, few studies have evaluated the risk factors of these complications after hepatolithiasis treatment . Moreover, previous studies have only evaluated the risk factors of recurrent cholangitis after initial surgical and/or endoscopic treatment (5, 6). Furthermore, the results of previous articles are inadequate to identify the risk factors responsible for hepatobiliary complications after treatment . Therefore, we sought to identify the risk factors and long - term hepatobiliary outcomes with respect to recurrent cholangitis, liver abscess, secondary biliary cirrhosis, and cholangiocarcinoma, in patients with hepatolithiasis after treatment . The medical records of 260 patients with hepatolithiasis that were surgically and/or endoscopically managed at our institution between december 1996 and august 2005 were retrospectively reviewed . We excluded 175 patients due to concurrent malignancy, underlying biliary cirrhosis, a congenital anomaly, incomplete medical records, or postoperative mortality (table 1). Hepatolithiasis was confirmed by abdominal ultrasonography, computed tomography, endoscopic retrograde cholangiopancreatography, magnetic resonance cholangiopancreatography, and/or surgery . Further examinations depended on the ultrasonographic findings, severity of associated symptoms, combined clinical problems, and the intended mode of treatment . When the presence of intrahepatic duct stones, a liver mass, or a liver abscess was suspected based on sonographic findings, further examinations, such as abdominal ct, magnetic resonance cholangiopancreatography, and/or biopsy, were undertaken . Definite indications for hepatectomy were stones localized in a unilateral lobe, bile duct stricture associated with stones, or atrophy of the affected liver segments or lobe . Hepatectomy was performed at the lobe with intrahepatic stones . However, if treatment provided by endoscopic methods was inadequate in patients with stones in both lobes, a surgery strategy was adopted . On the other hand, patients at high operative risk, including the elderly, and those that refused operative treatment were treated endoscopically . Follow - up was defined as time from date of discharge after initial treatment to the detection of subsequent cholangitis, liver abscess, secondary biliary cirrhosis, or cholangiocarcinoma . A residual stone was defined as a stone detected within 6 months by any diagnostic method . Bile duct stricture was defined as definite narrowing of bile duct documented by cholangiography, cholangioscopy, or surgery . Recurrent cholangitis was defined as a condition accompanied by presence of abdominal pain, fever, and/or jaundice without any other infection focus outside the hepatobiliary system requiring antibiotic administration . Univariate analysis of individual and total hepatobiliary complications was performed using the chi - square test, fisher's exact test, and the t - test . Were approved by institutional review board of inha university hospital (iuh - irb 13 - 1906). Univariate analysis of individual and total hepatobiliary complications was performed using the chi - square test, fisher's exact test, and the t - test . The study protocol and amendments were approved by institutional review board of inha university hospital (iuh - irb 13 - 1906). There were 21 men and 64 women, whose ages ranged from 37 to 83 yr (mean, 59.4 yr). Of the patients, 65 were surgically treated and 20 underwent percutaneous transhepatic cholangioscopic lithotripsy (ptcsl). Residual stone rates were 35% in patients treated with ptcsl (7/20) and 26.2% in patients treated surgically (17/65). Biliary stricture was found in 35 patients, and 29/35 patients were manipulated by endoscopic dilation or segmental hepatectomy . However, surgery was performed in patients with stones located in bilateral lobes and liver atrophy in only one lobe, to prevent cholangiocarcinoma . Similarly, partial segmentectomy was performed in patients with segmental atrophy of the liver and inadequate liver function . Recurrent cholangitis occurred in 14 (16.5%), liver abscess in 3 (3.5%), secondary biliary cirrhosis in 5 (5.9%), and cholangiocarcinoma in 2 (2.4%). Patients with recurrent cholangitis were treated by peroral transpapillary endoscopic lithotripsy (n=6), ptcsl (n=4), surgery (n=1), or systemic antibiotics only (n=3). Univariate analysis showed that the presences of bile duct stricture and residual stones were significant predictors of hepatobiliary complications (p = 0.001, p = 0.009, respectively). The rate of hepatobiliary complication was 34.3% in patients with a biliary stricture and 37.5% in patients with a residual stone . Age, sex, and treatment methods were not found to be risk factors of hepatobiliary complications (table 3). Multivariate analysis included age, sex, the presence of a residual stone, the presence of bile duct stricture, and treatment method . Age (hr, 1.046; ci, 1.006 - 1.089), bile duct stricture (hr, 4.894; ci, 1.295 - 18.495), and residual stone (hr, 3.482; ci, 1.214 - 9.981) were found to be independently correlated with the risk of developing a hepatobiliary complication (table 4). 1 demonstrates the cumulative risks of hepatobiliary complications according to the presence of bile duct stricture or a residual stone, respectively . Although hepatolithiasis is a benign condition, the clinical progression of the disease may lead to liver parenchymal destruction due to recurrent cholangitis (7). Furthermore, hepatolithiasis can subsequently result in biliary cirrhosis and even cholangiocarcinoma (8), which usually result from inadequate stone removal or stricture treatment (2). Residual stones are the most troublesome problem after treatment (9). In a previous study, the overall recurrence rate of hepatolithiasis and/or cholangitis in patients with a residual stone after initial treatment was 57% (39/69). On the other hand, the recurrence rate of hepatolithiasis and/or cholangitis in patients without a residual stone was only 17% (27/167). In addition, it has been reported that recurrent biliary symptoms were more common in patients with a residual stone than in patients without a residual stone (6). Another retrospective study of 193 patients with hepatolithiasis, who had been newly diagnosed and treated, reported similar results (4). In the study, cholangitis developed more commonly in patients with retained stones after hepatolithiasis treatment and cholangitis also developed more in patients with recurrent calculi in intrahepatic bile ducts than in patients without residual or recurrent stones (60% and 55% vs 29% and 9%, respectively). The results are similar to the present study, in which the complication rate in patients with a residual stone after hepatolithiasis treatment was 37.5% but the rate in patients without a residual stone was only 9.8% . Furthermore, multivariate analysis showed that the presence of a residual stone was independently related to the risk of developing a hepatobiliary complication . In our experience, hepatolithiasis patients who retained residual stones even after aggressive treatment often had a good prognosis without any hepatobiliary events during long - term follow - up . We speculate that the good prognosis in this subset of patients with a residual stone, was due to the tendency of residual stones to locate to the peripheries of intrahepatic ducts and not to migrate into main ducts . Nevertheless, the presence of a residual stone was found to be an independent risk factor of a long - term hepatobiliary complication . Intrahepatic biliary stricture is a major cause of treatment failure for intrahepatic stones and the main cause of stone recurrence . Biliary stricture often gives rise to bile stasis, cholangitis, and stone formation, and if the diseased ducts are not removed, the possibility of stone recurrence is high (2). One retrospective study reviewed 74 patients treated for hepatolithiasis . At 1- to 23-yr follow - ups, patients with a bile duct stricture were found to have a higher rate of incomplete clearance (30% [11/37] vs 5% [2/37]; p furthermore, the recurrence rate of hepatolithiasis was higher in patients with a bile duct stricture (69% [18/26] vs 37% [13/35]; p, biliary stricture was found to be associated with a recurrent hepatobiliary complication after treatment, and the rate of stone and/or cholangitis recurrence was greater in patients with an intrahepatic duct stricture than in those without (31% [41/133] vs 47% [16/34]; p = 0.075). Furthermore, multivariate analysis showed that an intrahepatic duct stricture was an independent risk factor of stone recurrence, cholangitis, and residual stones after treatment (4). In the present study, hepatobiliary complication rate was 34.3% in patients with a biliary stricture, and by multivariate analysis, bile duct stricture was independently correlated with the risk of developing a hepatobiliary complication (table 4). These findings show that complete stone clearance and the elimination of intrahepatic duct strictures, is necessary to prevent recurrent hepatobiliary complications in patients with hepatolithiasis . Stricture dilation with a balloon catheter has been reported to decrease the risk of recurrent cholangitis in hepatolithiasis concurrent with biliary stricture (10). However, biliary stricture has also been reported to be an independent risk factor of long - term hepatobiliary complications in patients with hepatolithiasis, regardless of stricture dilation (5). Generally, the prevalence of intrahepatic stones increases with age (11). However, we were unable to find any study that investigated the relations between recurrent hepatolithiasis and age . In the present study, patient age was found to be an independent risk factor for a long - term hepatobiliary complication . An age threshold was used to evaluate its influence on the rate of recurrent hepatobiliary complications . Univariate analysis showed that age was not a significant risk factor of a hepatobiliary complication, but cox proportional hazards analysis showed that age was significantly associated with a long - term hepatobiliary complication . A previous study concluded that hepatectomy is more effective than non - hepatectomy surgical treatment for hepatolithiasis (3). Hepatectomy seems to be the most definitive approach because it allows stone and biliary stricture removal simultaneously, and thus, reduces the risk of intrahepatic stone recurrence (12). However, other surgical methods, such as, choledochoenterostomy and t - tube drainage are available (13). Sometimes surgery may not be an option in those at high surgical risk who refuse surgery, or those that have undergone biliary surgery, or when stones are distributed in multiple segments (14). The endoscopic treatment of hepatolithiasis is considered relatively safe (11), but access is limited by strictures, ductal angulation, and the degree of stone impaction (12). The cholangioscopy was introduced to visualize stones, blood clots, air bubbles, and duct strictures and to improve stone clearance success rates (15). The first use of percutaneous transhepatic cholangioscopic lithotripsy for the treatment of hepatolithiasis was described in 1981, and it is now common used to remove intrahepatic duct stones non - surgically . However, almost 40% of cases of hepatolithiasis patients have intrahepatic bile duct strictures, which make stone extraction difficult (2). Furthermore, bile duct strictures prevent the definitive removal of sclerotic damage to intrahepatic biliary ducts, and this predisposes the recurrence of septic complications and the need for repeated treatments (16). In our study, treatment method was not found to be a significant risk factor of hepatobililary complications after initial treatment for hepatolithiasis by statistical analysis . However, this result could have been caused by selective bias, because when decisions were made regarding surgical and endoscopic methods, patient condition and location of stones in the biliary tract were considered . Thus, when stones were deemed not amenable to endoscopy, a surgical method was chosen . First, it is inherently limited by its retrospective nature and particularly by the absence of clear clinical information . Second, the study involved a relatively small number of patients, and it was performed at a single medical center, and thus, our results may not be representative of the general population . More specifically, the incidence of cholangiocarcinoma and the residual stone rate could have been affected by bias, as is suggested by a lower incidence of cholangiocarcinoma and a higher residual stone rate than have been previously reported . In conclusion, the present study shows the presence of a bile duct stricture is the strongest predictor of subsequent complications after initial treatment for hepatolithiasis . Furthermore, hepatobiliary complications, such as, recurrent cholangitis, liver abscess, secondary biliary cirrhosis, and cholangiocarcinoma may occur frequently even after multidisciplinary treatment . Therefore, close observation is required especially in hepatolithiasis concomitant with bile duct stricture or residual stones, and in the elderly, even after constructive treatment.
Non - st - segment elevation acute coronary syndromes (nste - acs) are the most frequent manifestation of acute coronary syndromes (acs). Its morbidity and mortality stays high and even equal to those of patients with st - segment elevation myocardial infarction (stemi) during long - term follow - up.1)2) current guidelines2)3) propose risk stratification for tailoring treatment in patients with nste - acs . In patients with intermediate- to very high - risk nste - acs, routine invasive diagnostics and treatments are recommended . Multivessel coronary disease (mvd), a leading pathological foundation of intermediate to very high risk clinical manifestation, accounts for approximately 3040% nste - acs cohorts overall and is usually treated with invasive interventions.4) the american college of cardiology / american heart association guidelines provide a class iib recommendation that multivessel percutaneous coronary intervention (pci), in contrast to culprit - only pci, might be reasonable in nste - acs patients undergoing pci (level of evidence: b).3) this recommendation is based on reports of studies suggesting that multivessel pci is superior to culprit vessel only pci in terms of repeat revascularization.5)6)7)8) however, there is still uncertainty as to whether non - culprit lesions should be treated at the time of culprit - lesion pci for nste - acs.3) in the present study, we sought to examine clinical outcomes of " one - time " versus staged multivessel stenting in intermediate to very high - risk nste - acs patients with mvd . Between november 2008 and november 2012, a total of 12047 unselected patients who had undergone a pci were prospectively registered in the pci database of general hospital of shenyang military region . The database contained comprehensive information including clinical and angiographic characteristics, treatment strategies and clinical outcomes . Eligible patients had at least one intermediate to very high risk criteria with indication for invasive management defined by the 2015 european society of cardiology (esc) guidelines for the management of nste - acs2); 3 types of very high - risk criteria include recurrent or ongoing chest pain refractory to medical treatment, and recurrent dynamic st - t wave changes particularly with intermittent st - elevation and acute heart failure; 3 types of high - risk criteria include rise or fall in cardiac troponin compatible with myocardial infarction (mi), dynamic st- or t - wave changes and global registry of acute coronary events (grace) score> 140; 7 intermediate - risk criteria include diabetes mellitus, renal insufficiency (estimated glomerular filtration rate [egfr] <60 ml / min/1.73 m), left ventricular ejection fraction (lvef) <40% or congestive heart failure, early post - infarction angina, prior pci, prior coronary artery bypass graft (cabg) and grace risk score>109 and <140 . Exclusion criteria were patients who had chronic total occlusion; patients who had procedural failure (i.e., technical failure), including staged pci patients who had procedural failure during the index pci and scheduled for staging; patients who had cardiac shock, haemodynamic instability, mechanical complications of mi or malignant ventricular arrhythmia; patients who had renal dialysis or a egfr <30 ml / min/1.73 m; staged pci patients who had any major complication during the index procedure; patients who had a planned staged pci> 60 days . Our final population included 1531 patients from the database having nste - acs with multivessel pci (fig . All patients were given oral loading doses of aspirin (300 mg) and clopidogrel (300 - 600 mg) prior to pci, unless they had already received antiplatelet medication . Interventional procedures were performed according to standard techniques and interventional strategies rested on the operators . The culprit lesion was identified by operators usually based on each patient's electrocardiogram, angiographic imaging, echocardiogram and, if available, intravascular ultrasound (ivus) and optical coherence tomography (oct). A lesion was considered a culprit on angiography if at least two of the following morphological features suggestive of acute plaque rupture were present: intraluminal filling defects consistent with thrombus, plaque ulceration, plaque irregularity, dissection or impaired flow.2)9)10)11) after the procedure, the use of aspirin lifelong was advised and clopidogrel was prescribed for 12 months . The study was approved by the hospital ethics committee and all patients gave written informed consent . Clinical follow - up was performed via telephone or at an outpatient visit at 30 days, 6 months, and 12 months after the index procedure and annually until 3 years after the index procedure . Follow - up angiography was recommended to all patients 6 to 12 months after the index procedure, and repeat revascularization was performed, if clinically indicated . The primary outcome was the incidence of major adverse cardiac events (mace), defined as the composite of cardiac death, mi and target vessel revascularization (tvr) during 3-year follow - up . Secondary outcomes included mace components, the composite of cardiac death or mi, definite / probable stent thrombosis, and any repeat revascularization . Mvd was defined as the presence of a significant atherosclerotic coronary artery stenosis (70% diameter stenosis) or a 50% stenosis of the left main coronary artery (left main disease) with additional significant stenosis (70% diameter stenosis) of at least one other coronary artery assessed visually during coronary angiography.12) staged pci is defined as the planned pci of non - infarct vessel(s) within 60 days of the index pci . Egfr was calculated from serum creatinine (scr) concentrations using the modified glomerular filtration rate estimating the equation for chinese patients with chronic kidney disease: egfr (ml / min/1.73 m)=175(scr)(age)(0.79 if patient is female).13) anemia was defined as hemoglobin less than 12 g / dl for men or less than 11 g / dl for women.14) early invasive intervention was defined as a coronary angiography performed within 24 h of hospital admission.2) delayed invasive intervention was defined as a coronary angiography performed more than 24 h of hospital admission . Technical success was defined as the ability to cross an occluded or stenosed segment and successfully open the artery (restoration of thrombolysis in myocardial infarction [timi] flow grade 3) with a residual stenosis 30% by visual analysis . Cardiac death was defined as death that could not be attributed to a non - cardiac etiology . Mi was defined as third universal definition of mi presented by the third global mi task force.15) tvr was determined as any repeated pci or cabg to treat a previously stented vessel . Complete revascularization was defined when no visually estimated stenosis 50% for the left main and no stenosis 70% for other major arteries and/or their major branches at discharge . Stent thrombosis was classified as definite and probable according to definitions proposed by the academic research consortium.16) continuous variables were expressed as meanstandard deviation (sd), and categorical variables were expressed as number and/or percentages . For group comparisons, pearson chi - square test or fisher's exact test was used for categorical variables . Student's unpaired t - test or the mann - whitney rank - sum test, as appropriate, was used for continuous variables . To minimize the influence of confounders on outcome, we used propensity score matching analysis . Patients who underwent staged pci were matched in a 1:1 ratio with patients who underwent a " one - time " pci using the nearest neighbor matching, which were based on all available variables listed in tables 1 and 2 except grace score, ivus used, oct used, contrast volume, length of hospital, medication at discharge and dual antiplatelet therapy (dapt) duration . A difference of <10% was regarded as acceptable . Time to event data with estimated event rates calculated by kaplan - meier method were compared with the log - rank test . In addition, we performed kaplan - meier analyses at the landmark periods of 0 to 90 days and 90 days to 3 years to evaluate the effect of revascularization strategy on clinical outcomes at different time periods . Between november 2008 and november 2012, a total of 12047 unselected patients who had undergone a pci were prospectively registered in the pci database of general hospital of shenyang military region . The database contained comprehensive information including clinical and angiographic characteristics, treatment strategies and clinical outcomes . Eligible patients had at least one intermediate to very high risk criteria with indication for invasive management defined by the 2015 european society of cardiology (esc) guidelines for the management of nste - acs2); 3 types of very high - risk criteria include recurrent or ongoing chest pain refractory to medical treatment, and recurrent dynamic st - t wave changes particularly with intermittent st - elevation and acute heart failure; 3 types of high - risk criteria include rise or fall in cardiac troponin compatible with myocardial infarction (mi), dynamic st- or t - wave changes and global registry of acute coronary events (grace) score> 140; 7 intermediate - risk criteria include diabetes mellitus, renal insufficiency (estimated glomerular filtration rate [egfr] <60 ml / min/1.73 m), left ventricular ejection fraction (lvef) <40% or congestive heart failure, early post - infarction angina, prior pci, prior coronary artery bypass graft (cabg) and grace risk score>109 and <140 . Exclusion criteria were patients who had chronic total occlusion; patients who had procedural failure (i.e., technical failure), including staged pci patients who had procedural failure during the index pci and scheduled for staging; patients who had cardiac shock, haemodynamic instability, mechanical complications of mi or malignant ventricular arrhythmia; patients who had renal dialysis or a egfr <30 ml / min/1.73 m; staged pci patients who had any major complication during the index procedure; patients who had a planned staged pci> 60 days . Our final population included 1531 patients from the database having nste - acs with multivessel pci (fig . All patients were given oral loading doses of aspirin (300 mg) and clopidogrel (300 - 600 mg) prior to pci, unless they had already received antiplatelet medication . Interventional procedures were performed according to standard techniques and interventional strategies rested on the operators . The culprit lesion was identified by operators usually based on each patient's electrocardiogram, angiographic imaging, echocardiogram and, if available, intravascular ultrasound (ivus) and optical coherence tomography (oct). A lesion was considered a culprit on angiography if at least two of the following morphological features suggestive of acute plaque rupture were present: intraluminal filling defects consistent with thrombus, plaque ulceration, plaque irregularity, dissection or impaired flow.2)9)10)11) after the procedure, the use of aspirin lifelong was advised and clopidogrel was prescribed for 12 months . The study was approved by the hospital ethics committee and all patients gave written informed consent . Clinical follow - up was performed via telephone or at an outpatient visit at 30 days, 6 months, and 12 months after the index procedure and annually until 3 years after the index procedure . Follow - up angiography was recommended to all patients 6 to 12 months after the index procedure, and repeat revascularization was performed, if clinically indicated . The primary outcome was the incidence of major adverse cardiac events (mace), defined as the composite of cardiac death, mi and target vessel revascularization (tvr) during 3-year follow - up . Secondary outcomes included mace components, the composite of cardiac death or mi, definite / probable stent thrombosis, and any repeat revascularization . Mvd was defined as the presence of a significant atherosclerotic coronary artery stenosis (70% diameter stenosis) or a 50% stenosis of the left main coronary artery (left main disease) with additional significant stenosis (70% diameter stenosis) of at least one other coronary artery assessed visually during coronary angiography.12) staged pci is defined as the planned pci of non - infarct vessel(s) within 60 days of the index pci . Egfr was calculated from serum creatinine (scr) concentrations using the modified glomerular filtration rate estimating the equation for chinese patients with chronic kidney disease: egfr (ml / min/1.73 m)=175(scr)(age)(0.79 if patient is female).13) anemia was defined as hemoglobin less than 12 g / dl for men or less than 11 g / dl for women.14) early invasive intervention was defined as a coronary angiography performed within 24 h of hospital admission.2) delayed invasive intervention was defined as a coronary angiography performed more than 24 h of hospital admission . Technical success was defined as the ability to cross an occluded or stenosed segment and successfully open the artery (restoration of thrombolysis in myocardial infarction [timi] flow grade 3) with a residual stenosis 30% by visual analysis . Cardiac death was defined as death that could not be attributed to a non - cardiac etiology . Mi was defined as third universal definition of mi presented by the third global mi task force.15) tvr was determined as any repeated pci or cabg to treat a previously stented vessel . Complete revascularization was defined when no visually estimated stenosis 50% for the left main and no stenosis 70% for other major arteries and/or their major branches at discharge . Stent thrombosis was classified as definite and probable according to definitions proposed by the academic research consortium.16) continuous variables were expressed as meanstandard deviation (sd), and categorical variables were expressed as number and/or percentages . For group comparisons, pearson chi - square test or fisher's exact test was used for categorical variables . Student's unpaired t - test or the mann - whitney rank - sum test, as appropriate, was used for continuous variables . To minimize the influence of confounders on outcome, we used propensity score matching analysis . Patients who underwent staged pci were matched in a 1:1 ratio with patients who underwent a " one - time " pci using the nearest neighbor matching, which were based on all available variables listed in tables 1 and 2 except grace score, ivus used, oct used, contrast volume, length of hospital, medication at discharge and dual antiplatelet therapy (dapt) duration . A difference of <time to event data with estimated event rates calculated by kaplan - meier method were compared with the log - rank test . In addition, we performed kaplan - meier analyses at the landmark periods of 0 to 90 days and 90 days to 3 years to evaluate the effect of revascularization strategy on clinical outcomes at different time periods . " One - time " pci was performed in 56.1% (859/1531), and the remaining 43.9% (672/1531) had staged pci (of these, 80.7% [542/672] had staged non - culprit intervention during the same hospitalization and 19.3% [130/672] had planned staged non - culprit procedures after hospital discharge). The median delay of the staged pci was 5 days (interquartile rang [iqr], 3 - 9 days). As noted in tables 1 and 2, male, previous mi, triple - vessel disease, and current smoking were associated with more staging . The volume of contrast media utilized during the index procedure in the staged pci group was smaller (200 ml [iqr, 150 - 220 ml] vs. 200 ml [iqr, 200 - 300 ml], p<0.001) though the total volume utilized in the initial procedure plus staged procedure was greater (400 ml [iqr, 310 - 500 ml] vs. 200 ml [iqr, 200 - 300 ml], p<0.001). Most patients took dual antiplatelet therapy (dapt), which was consistent with the standard recommendation . After propensity matching, there were no statistically significant differences in preselected variables between the two groups . 3a, mace occurred in 132 patients (20.8%) in the staged group and 162 patients (19.7%) in the " one - time " group during 3-year follow - up (p=0.608). Rates of each component of 3-year cumulative mace were similar between both groups . The estimated 3-year composite rate of cardiac death or mi was 7.1% in the staged pci group compared with 9.1% in the " one - time " pci group (p=0.129). In addition, no significant differences in the 3-year rates of any revascularization (22.3% vs. 18.6%, p= 0.083) and definite / probable stent thrombosis (1.3% vs. 1.9%, p=0.295) were observed . At the 90-day landmark analysis (tables 3 and 4, fig . 2c and fig . 3c), no significant differences in terms of mace, its individual components, any revascularization and definite / probable stent thrombosis were observed at the landmark period of 0 to 90 days and 90 days to 3 years . After generating a propensity score, 420 of the 672 patients who underwent staged pci were matched with a patient, respectively, who underwent a " one - time " pci . There were no differences in preselected variables for the propensity matched subjects (table 1, 2). 3b, at 3 years, there were no differences in mace (18.9% vs. 21.8%, p=0.249); whereas there was a significantly lower incidence of cardiac death or mi (7.0% vs. 11.1%, p=0.033). The risk for cardiac death in the staged pci group tended to be lower (2.5% vs. 4.8%, p=0.067). Other clinical outcomes including mi (5.0% vs. 7.7%, p=0.162), tvr (14.9% vs. 14.5%, p=0.971), any revascularization (20.3% vs. 20.4%, p=0.888), and definite / probable stent thrombosis (1.2% vs. 2.2%, p=0.280) were not significantly different between the two study groups . The results of the 90-day landmark analysis for propensity score matched patients were shown in tables 3 and 4, fig . The staged pci group showed a lower 90-day incidence of mace (1.2% vs. 3.3%, p=0.037), mainly due to a lower composite rate of cardiac death or mi (0.7% vs. 2.6%, p=0.031). The 90-day rates of mi did not differ significantly between the 2 study groups, but it presented a trend in favor of staged pci (0.7% vs. 2.1%, p=0.081). For the 90-day to 3-year follow - up period, both the incidences of mace (17.9% vs. 19.1%, p=0.641) and the composite of cardiac death or mi (6.3% vs. 8.7%, p=0.191) were similar between the two groups . Cardiac death, tvr, any revascularization and definite / probable stent thrombosis did not differ significangtly between the two groups at the landmark period of 0 to 90 days and 90 days to 3 years . " One - time " pci was performed in 56.1% (859/1531), and the remaining 43.9% (672/1531) had staged pci (of these, 80.7% [542/672] had staged non - culprit intervention during the same hospitalization and 19.3% [130/672] had planned staged non - culprit procedures after hospital discharge). The median delay of the staged pci was 5 days (interquartile rang [iqr], 3 - 9 days). As noted in tables 1 and 2, male, previous mi, triple - vessel disease, and current smoking were associated with more staging . The volume of contrast media utilized during the index procedure in the staged pci group was smaller (200 ml [iqr, 150 - 220 ml] vs. 200 ml [iqr, 200 - 300 ml], p<0.001) though the total volume utilized in the initial procedure plus staged procedure was greater (400 ml [iqr, 310 - 500 ml] vs. 200 ml [iqr, 200 - 300 ml], p<0.001). Most patients took dual antiplatelet therapy (dapt), which was consistent with the standard recommendation . After propensity matching, there were no statistically significant differences in preselected variables between the two groups . 2a and fig . 3a, mace occurred in 132 patients (20.8%) in the staged group and 162 patients (19.7%) in the " one - time " group during 3-year follow - up (p=0.608). Rates of each component of 3-year cumulative mace were similar between both groups . The estimated 3-year composite rate of cardiac death or mi was 7.1% in the staged pci group compared with 9.1% in the " one - time " pci group (p=0.129). In addition, no significant differences in the 3-year rates of any revascularization (22.3% vs. 18.6%, p= 0.083) and definite / probable stent thrombosis (1.3% vs. 1.9%, p=0.295) were observed . At the 90-day landmark analysis (tables 3 and 4, fig . 2c and fig . 3c), no significant differences in terms of mace, its individual components, any revascularization and definite / probable stent thrombosis were observed at the landmark period of 0 to 90 days and 90 days to 3 years . After generating a propensity score, 420 of the 672 patients who underwent staged pci were matched with a patient, respectively, who underwent a " one - time " pci . There were no differences in preselected variables for the propensity matched subjects (table 1, 2). 3b, at 3 years, there were no differences in mace (18.9% vs. 21.8%, p=0.249); whereas there was a significantly lower incidence of cardiac death or mi (7.0% vs. 11.1%, p=0.033). The risk for cardiac death in the staged pci group tended to be lower (2.5% vs. 4.8%, p=0.067). Other clinical outcomes including mi (5.0% vs. 7.7%, p=0.162), tvr (14.9% vs. 14.5%, p=0.971), any revascularization (20.3% vs. 20.4%, p=0.888), and definite / probable stent thrombosis (1.2% vs. 2.2%, p=0.280) were not significantly different between the two study groups . The results of the 90-day landmark analysis for propensity score matched patients were shown in tables 3 and 4, fig . The staged pci group showed a lower 90-day incidence of mace (1.2% vs. 3.3%, p=0.037), mainly due to a lower composite rate of cardiac death or mi (0.7% vs. 2.6%, p=0.031). The 90-day rates of mi did not differ significantly between the 2 study groups, but it presented a trend in favor of staged pci (0.7% vs. 2.1%, p=0.081). For the 90-day to 3-year follow - up period, both the incidences of mace (17.9% vs. 19.1%, p=0.641) and the composite of cardiac death or mi (6.3% vs. 8.7%, p=0.191) were similar between the two groups . Cardiac death, tvr, any revascularization and definite / probable stent thrombosis did not differ significangtly between the two groups at the landmark period of 0 to 90 days and 90 days to 3 years . To our knowledge, this is the first study to examine the efficacy of staged versus " one - time " approach in intermediate to very high - risk nste - acs patients with multivessel pci . Main findings of this study were as follow: in intermediate to very high - risk nste - acs patients with multivessel disease, staged pci was associated with a lower composite rate of cardiac death or mi compared with " one - time " pci strategy . This benefit of staged pci was more apparent in the early period after procedures, rather than middle - late period . Clinicians are often faced with the decision of whether to treat the nonculprit lesions during the index procedure or to treat it at a later time.17) hannan et al . Compared the " one - time " complete revascularization in the index admission versus pci of the culprit lesion only with staged non - culprit pci for complete revascularization in a subsequent admission among patients with nste - acs and mvd.18) at 3 years, no significant difference in all - cause mortality was observed between the two groups . However, the staged pci group did not include the patients who underwent a staged intervention during the same hospitalization . Furthermore, it is yet unknown whether it was different in all - cause mortality between the two groups after exclusions of low risk patients . In the current observational study, the superiority of staged pci strategy over " one - time " pci strategy in terms of the composite of cardiac death or mi was presented, especially in the early period after pci . First, nste - acs patients are in a heightened thrombotic and inflammatory state.19)20)21)22) " one - time " multivessel pci can lead to prolongation of procedure time, exposure to a higher radiation dose23) and larger volume of contrast agents during the index procedure compared to staged revascularization . This may result in an increased number of complications such as periprocedural mi or acute kidney injury as well as stent thrombosis.24)25) moreover, patients with high / intermediate risks of nste - acs are often in a poor clinical state or have concomitant comorbidities . Pci on the culprit lesion first and staged non - culprit pci at a later date with optimal medical therapy usually results in the patient being stable and also allows clinicians to reassess the patient's clinical and angiographic states . Risk stratification is essential for the clinical decision - making process in nste - acs patients . With respect to outcomes, periprocedural complications of intervention, as well as the long - term ischemic risk, remain higher in high / intermediate risk nste - acs than in low - risk nste - acs patients.26)27)28)29)30) when choosing the optimal treatment strategy in the individual patient with nste - acs and mvd, general patient condition and concomitant comorbidities have to be taken into account . However, the optimal strategy for the management of nste - acs patients with poor clinical presentation and multivessel disease has not been well established . Therefore, our patient inclusion criteria, which were based on esc guidelines, selected nste - acs patients with intermediate- to very high - risk features . The decisions for one - time vs. staged pci were not based on a randomization but at physicians' discretion, which resulted in obvious confounding and selection bias . Although we analyzed by adjusting many possible confounding factors, unmeasured confounding or selection bias might have influenced our findings . Second, in this study, all patients underwent pci in the setting of antiplatelet therapy with clopidogrel . Third, the relatively high frequency of angiography during the period between 6 to 12 months after the procedure could bias the mace, especially tvr . However, this bias was limited, because the two groups had similar frequencies of follow - up angiography and because repeat intervention was guided by either recurrent angina or signs of ischemia that had progressed angiographically, even without angina based on invasive or noninvasive testing . Fourth, our data were collected before current practice guidelines2) were published . Fifth, given the low absolute number of events at 90-day follow up in the propensity score matched cohort, our study was not powered to detect significant differences in cardiac death, mi, tvr and stent thrombosis at 90 days . Last, considering follow - up angiography and routine cardiac biomarkers surveillance was not mandatory, therefore the incidence of mi might have been underestimated . In intermediate to very high - risk nste - acs patients with multivessel disease, staged pci is superior to " one - time " pci in terms of the composite of cardiac death or mi . Our findings require further confirmation by randomized trial . In intermediate to very high - risk nste - acs patients with multivessel disease, staged pci is superior to " one - time " pci in terms of the composite of cardiac death or mi . Our findings require further confirmation by randomized trial.
The hira data tables were composed of five main tables (general specification, healthcare services, diagnosis information, prescriptions, and general information about the medical institutions). The disease codes were based on the korean standard classification of disease (kcd). Specifications of the computer that was used to analyze the data were as follows: central processing unit (cpu), i3 - 2120 3.30 ghz (intel, santa clara, ca, usa); 32 gb main memory; operating system (os), ubuntu 14.04.3 long - term support (lts); and r 3.2.2 analysis software . The sample data contained 22,344,536 claims in 1,361,717 patients (selection probability, 0.03; sampling weight, 33.3). In the year 2013, 163,372 pathologic examinations were performed in 103,528 patients (45,897 men and 57,631 women). The mean and median ages of the patients were 51.8 and 53 years, respectively . The pathologic examination data and medical institutions according to administrative district are summarized in table 2 . The total numbers of medical institutions according to administrative district are summarized in appendix 1 . In total, 43.5% of the tertiary hospitals (10/23), 15.6% of the general hospitals (48/307), and 23.8% of the other institutions (12,090/50,868) were located in seoul . In addition, 32.9% of the surgical pathologic examinations (53,711/163,372) were requested in medical institutions located in seoul . Almost all medical and dental departments requested pathologic examinations (table 3). Internal medicine (74,232, 45.4%), general surgery (34,808, 21.3%), obstetrics and gynecology (19,382, 11.9%), and urology (9,951, 6.1%) were the most common medical departments (84.7%) requesting pathology examinations . A small subset (38/17,892, 0.2%) of immunohistochemical (ihc) stains were not interpreted by qualified doctors (not claimed as c5575006) (data not shown). Claims for acetylcholinesterase (c5564) and chloroacetate esterase (c5565) examinations were not found in our data . Among pathologic examination - associated claims, 162,002 examinations (99.2%) had proper claim codes (examination codes 09 and sub - code 01 (performed in their own institutions) or 02 (performed in outside institutions). Among pathologic examinations, 65.8% were performed in tertiary or general hospitals (39,385 and 67,225 cases, respectively) (table 4). Almost all examinations claimed by tertiary hospitals were performed in their institute (39,349/39,385, 99.9%), and 85% of examinations claimed by general hospitals were performed (57,163/67,225) in their institute . The other medical institutions claimed considerable pathologic examinations performed by outside services (52,214/55,392, 94.3%). According to the claims, 76,016 pathologic examinations (44.7%) were performed due to malignancy (disease code c) or non - malignant tumorous conditions (disease code d00-d48) (table 5). Pathologic examination of biopsy specimens was performed more frequently in non - tumorous conditions (42,661/61,875, 68.9%). In total, 509 esds were performed in 490 patients (341 males and 149 females) in 109 medical institutions . The median age of the patients was 66 years (age, 29 to 89 years; 1st quantile, 58 years; 3rd quantile, 71.75 years). The majority of patients (n=472) underwent one esd; 17 patients underwent two esds (10 patients underwent simultaneous esd, while seven patients underwent esds at different times), and one patient underwent three esds (two esds at the same time). The disease codes noted when patients underwent esd were as follows: c16 (malignant neoplasm of stomach), 213 patients (43.5%); non - c16, 277 patients (56.5%); d00.2 (carcinoma in situ of stomach), 17 patients; d13.1 (benign neoplasm of stomach), 226 patients; d13.9 (benign neoplasm of ill - defined site within the digestive system), one patient; and other, 33 patients . Disease codes changed after the esd in 10.5% of the non - c16 patients (29/277): d00.2 to c16, seven patients; d13.1 to d00.2, three patients; d13.1 to c16, 18 patients; and d13.9 to c16, one patient (table 6). Twenty - one patients (21/490, 4.3%) underwent gastrectomy following esd, and all of these patients underwent esd for only once . Fifteen patients received surgery at the same medical institution where esd was performed, and six patients received surgery at different medical institutions . The mean time between esd and surgery was 44 days . For further analysis, 472 patients who underwent esd for only once were selected (table 7) (c16, 202 patients; d00.2, 17 patients; d13.1, 220 patients; d13.9, one patient; other, 32 patients). In total, 70.0% of these pathologic examinations (329/472) were requested for histologic mapping (c5508). Ihc studies were performed in 22.5% of esds (106/472). Approximately one - third of the gastric cancer specimens (66/202, 32.7%) and 15.0% of the gastric benign neoplasm specimens (33/220) were subject to ihc studies . In the year 2013, 163,372 pathologic examinations were performed in 103,528 patients (45,897 men and 57,631 women). The mean and median ages of the patients were 51.8 and 53 years, respectively . The pathologic examination data and medical institutions according to administrative district are summarized in table 2 . The total numbers of medical institutions according to administrative district are summarized in appendix 1 . In total, 43.5% of the tertiary hospitals (10/23), 15.6% of the general hospitals (48/307), and 23.8% of the other institutions (12,090/50,868) were located in seoul . In addition, 32.9% of the surgical pathologic examinations (53,711/163,372) were requested in medical institutions located in seoul . Almost all medical and dental departments requested pathologic examinations (table 3). Internal medicine (74,232, 45.4%), general surgery (34,808, 21.3%), obstetrics and gynecology (19,382, 11.9%), and urology (9,951, 6.1%) were the most common medical departments (84.7%) requesting pathology examinations . A small subset (38/17,892, 0.2%) of immunohistochemical (ihc) stains were not interpreted by qualified doctors (not claimed as c5575006) (data not shown). Claims for acetylcholinesterase (c5564) and chloroacetate esterase (c5565) examinations were not found in our data . Among pathologic examination - associated claims, 162,002 examinations (99.2%) had proper claim codes (examination codes 09 and sub - code 01 (performed in their own institutions) or 02 (performed in outside institutions). Among pathologic examinations, 65.8% were performed in tertiary or general hospitals (39,385 and 67,225 cases, respectively) (table 4). Almost all examinations claimed by tertiary hospitals were performed in their institute (39,349/39,385, 99.9%), and 85% of examinations claimed by general hospitals were performed (57,163/67,225) in their institute . The other medical institutions claimed considerable pathologic examinations performed by outside services (52,214/55,392, 94.3%). According to the claims, 76,016 pathologic examinations (44.7%) were performed due to malignancy (disease code c) or non - malignant tumorous conditions (disease code d00-d48) (table 5). Pathologic examination of biopsy specimens was performed more frequently in non - tumorous conditions (42,661/61,875, 68.9%). In total, 509 esds were performed in 490 patients (341 males and 149 females) in 109 medical institutions . The median age of the patients was 66 years (age, 29 to 89 years; 1st quantile, 58 years; 3rd quantile, 71.75 years). The majority of patients (n=472) underwent one esd; 17 patients underwent two esds (10 patients underwent simultaneous esd, while seven patients underwent esds at different times), and one patient underwent three esds (two esds at the same time). The disease codes noted when patients underwent esd were as follows: c16 (malignant neoplasm of stomach), 213 patients (43.5%); non - c16, 277 patients (56.5%); d00.2 (carcinoma in situ of stomach), 17 patients; d13.1 (benign neoplasm of stomach), 226 patients; d13.9 (benign neoplasm of ill - defined site within the digestive system), one patient; and other, 33 patients . Disease codes changed after the esd in 10.5% of the non - c16 patients (29/277): d00.2 to c16, seven patients; d13.1 to d00.2, three patients; d13.1 to c16, 18 patients; and d13.9 to c16, one patient (table 6). Twenty - one patients (21/490, 4.3%) underwent gastrectomy following esd, and all of these patients underwent esd for only once . Fifteen patients received surgery at the same medical institution where esd was performed, and six patients received surgery at different medical institutions . For further analysis, 472 patients who underwent esd for only once were selected (table 7) (c16, 202 patients; d00.2, 17 patients; d13.1, 220 patients; d13.9, one patient; other, 32 patients). In total, 70.0% of these pathologic examinations (329/472) were requested for histologic mapping (c5508). Ihc studies were performed in 22.5% of esds (106/472). Approximately one - third of the gastric cancer specimens (66/202, 32.7%) and 15.0% of the gastric benign neoplasm specimens (33/220) were subject to ihc studies . In this study, we examined nation - wide statistics regarding surgical pathologic examination . Considering sampling weight, we estimate that 5,440,288 (163,37233.3) pathologic examinations were performed in korea in 2013 . We also surveyed pathologic examinations according to administrative district, requesting department, type of medical institutions, and patient conditions . These data will be useful for future planning and allocation of resources in the field of pathology and for the korean society of pathologists . There have been several reports regarding diagnostic discrepancies between endoscopic forceps biopsy and esd, as well as between esd and surgery . Recently, two large, single - center, retrospective studies revealed that 22.8% (465/2,041) and 31.7% (587/1,850) of cases changed diagnosis after endoscopic resection in asan medical center (seoul, korea) and samsung medical center (seoul, korea), respectively . Our hira - nps data revealed that only 10.9% of nation - wide cases experienced a change of diagnosis after esd . Hira - nps data does not include detailed pathologic diagnosis information such as tumor size, tumor differentiations, dysplastic degrees, etc . Shin et al . Reported that complete resection rates were not different according to absolute or expanded esd indications, though their data were not hira data . Although hira - nps data was not available for a sufficient number of esd patients (509 esds in 490 patients) to allow for an accurate assessment, and only limited clinicopathological information was available, the problems associated with the relatively few number of patients can be overcome through further research using the raw hira data . Histologic mapping of esd specimens is recommended by the gastrointestinal pathology study group of the korean society of pathologists . However, 30.2% (143/472) of esd specimens were not claimed as c5508 because precise histologic diagnosis of the esd specimen is essential to treat patients, we suggest that nationwide surveys be conducted in order to assure quality of pathologic examination of esd specimens . Ihc staining was performed in 22.5% (106/472) of esd cases . Unlike tissue immunofluorescent microscopic examinations or enzyme histochemistries further analysis using hira raw data will be needed to decrease the statistical errors and bias and to evaluate changes over time . During our analysis, we experienced many challenges; therefore, we provide advice and guidance for other researchers who would like to analyze the hira database . (1) it is essential to understand the framework of the hira database . The hira data is intended for insurance claims and not for research . (2) sample data are not appropriate for analyses requiring long - term follow - up . (3) some claim codes have sub - codes (in most cases, additional charges by experts). (4) disease codes and claims are not always accurate . (5) the analysis system should have at least 32 gb of main memory . (6) it is helpful to create relatively small data tables to decrease operation time . In the usual setting, r should use only one cpu core during calculation.
Sexual systems in angiosperms range from hermaphroditism (monomorphic populations of plants with bisexual flowers) to dioecy (dimorphic populations of male and female individuals) and include almost all imaginable combinations and gradations (table 1; [1, 2] and references therein). Such remarkable diversity of sexual systems has perplexed naturalists and evolutionary biologists for a long time [36]. The evolution of dioecy from a hermaphroditic ancestor has been particularly difficult to understand because the invasion and maintenance of unisexual mutants in a population of hermaphrodites require that the loss of fitness resulting from the loss of one sexual function be compensated by increased fitness gains through the remaining sexual function of the unisexual mutant [7, 8]. This requirement seems very restrictive, and therefore considerable effort has been devoted towards understanding the conditions under which dioecy can evolve [5, 818]. In contrast, the evolution of sexually dimorphic traits following the evolution of dioecy (successful establishment of only two reciprocal unisexual morphs in a population) has received less attention . Consequently, our current understanding of the evolution of sex - related traits ultimately leading to morphological or physiological differences between unisexual morphs (i.e., sexual dimorphism or secondary sexual traits) is still limited, despite recent advances and excellent syntheses on the topic [5, 6, 1921]. This paper focuses on one set of traits subject to becoming sexually dimorphic upon the evolution of dioecy: those traits that provide plants with defence against herbivores . Sex - biased herbivory may be one of the selective pressures conducive to the evolution of dioecy, and it can also be a consequence of sex - specific selection on patterns of resource allocation in dioecious species . Considering only the gynodioecy pathway of the evolution of dioecy, we can think of three possible scenarios regarding the role of herbivory in each of the two steps involved in this pathway (figure 1). The first step in the gynodioecy pathway to dioecy is the successful establishment of females (male - sterile mutants) in a population of hermaphrodites, thus resulting in a gynodioecious population . As mentioned above, this step requires that females compensate for the fitness loss incurred with the loss of the male function . The reallocation of resources freed from the male function towards defence may contribute towards fitness compensation if increased defence results in greater fitness for the females . Increased defence may result in lower herbivore damage on females than on hermaphrodites (figure 1, path b). Defence may be achieved through resistance: traits that reduce the rate of herbivore attack such as low nutritional content of tissues (particularly, n content), secondary metabolites, trichomes, cutin, waxy cuticles, lignin, and volatiles that attract natural enemies of herbivores; and also through tolerance: traits that mitigate the negative effects of damage on fitness, including higher or lower growth rates, mobilization of stored resources, and activation of apical meristems . If females reallocate resources to tolerance traits, they could be the morph with greater herbivore damage (figure 1, path c). The second step in the gynodioecy pathway to dioecy is the successful establishment of male individuals (female - sterile mutants) in a gynodioecious population followed by the loss of the hermaphroditic morph, thus resulting in a dioecious population . Upon the evolution of two unisexual morphs, defensive traits may evolve differently in each sex and eventually become sex linked [8486]. The particular way in which defensive traits diverge between sexes will depend on the costs and benefits derived from the specific pattern of resource allocation to growth, reproduction, and defence in each sex . Currently, it is thought that females generally evolve greater resistance than males (see the following; figure 1, path b). Therefore, we will not delve into morph - biased herbivory in gynodioecious species, which would be the topic of a different essay . However, we do recognize that sex - biased herbivory indicative of sexual dimorphism in resistance against herbivores is likely related to morph - biased herbivory in the ancestral gynodioecious population from which it evolved (figure 1, path b - b). Thus, male - biased herbivory may have its origin in a gynodioecious population where hermaphrodites (functionally, the male morph) bear greater levels of herbivory than females . The above view for the origin of greater resistance against herbivores in females is based directly on the principle of allocation: resources freed from the male function are used for the female function, growth, and defence . In contrast to this view, the finding of male - biased herbivory in dioecious populations has been explained on the basis of sex - specific selection of resistance traits, where the main difference between sexes that drives the sex - specific selection is the cost of reproduction . In this alternative view, female individuals of dioecious species are expected to have lower herbivory levels than males because the higher cost of reproduction of females confers a selective advantage to females with traits that reduce herbivore attack . The logic of this argument is as follows: since females invest more in reproduction than males, they are left with a smaller pool of resources for growth and therefore must grow more slowly than males [20, 87]. According to the resource availability hypothesis, the fitness cost of losing tissue to herbivores is greater for plants that grow more slowly, thus favouring the evolution of increased defence against herbivores in slow - growing plants . Since females tend to grow more slowly, they should be better defended against herbivores than males . We must note that the argument for greater defence levels in females is usually understood in terms of resistance, but it could also be interpreted in terms of tolerance, in which case the predictions of sex - biased herbivory would be the opposite (figure 1, path c), as developed below . The first review of the empirical studies on the topic of sex - biased herbivory concluded that males are more likely than females to be preferentially used by herbivores and suggested that male - biased herbivory was widespread among dioecious species . The authors, however, recognized that sex - biased herbivory was by no means a unanimous finding across all the dioecious species examined to that date, and that the relative susceptibility of each sex to herbivory could be influenced, among other factors, by fluctuations in ecological tradeoffs between functions (rather than evolutionary changes in patterns of allocation), such as phenological changes in resource allocation to reproduction and growth [75, 89]. Therefore, the life stage at which damage measurements are taken can determine whether a study concludes that herbivory is sex biased or not . In addition, gren et al . Cautioned against publication bias, whereby studies that found differences between genders could be more likely to be published than those that did not; and taxonomic bias, an overabundance of studies from certain genera or families . In this instance, the taxonomic bias is correlated with an ecological bias for studies of temperate species, despite dioecy being more prevalent in tropical ecosystems . . Called for future studies that (1) examine the causes of differential palatability between males and females, (2) measure the fitness consequences of natural levels of herbivory in both sexes, and (3) determine whether herbivore pressure can actually cause adaptive changes in tissue palatability . In addition, they urged for broadening the taxonomical scope of the studies . In spite of such encouragement, there still is a paucity of studies that address these issues . More recently, sex - biased herbivory in dioecious species was tested by means of a meta - analysis of 33 studies encompassing 30 species, 19 of which were previously included in gren et al . However, they did not emphasize other shortcomings of the dataset and concluded that male - biased herbivory in dioecious species is a rule . Here we propose alternative evolutionary scenarios that could result in female - biased herbivory or lack of intersexual differences in herbivory levels . We invite the reader to reconsider the evidence for male - biased herbivory in dioecious plants and recommend a standard protocol for evolutionary - ecological studies of sex - biased herbivory in dioecious species that addresses the shortcomings listed below . We contend that taking for granted the generality of male - biased herbivory in dioecious species is hampering our progress in this field . While male - biased herbivory has been explained as a consequence of sex - specific selection of resistance determined by the cost of reproduction of each sex, few of the reviewed studies (table 2) are actually placed within such evolutionary context . J. lovett - doust and l. lovett - doust were the first to argue, citing charnov, that an evolutionary divergence between sexes in resource allocation patterns could result in sex - biased resistance against herbivory . Based their expectation of male - biased herbivory on sexual selection: it may be more advantageous for males to invest less in resistance and more in reproduction when exposed to a cyclic herbivore compared to a noncyclic herbivore because males will lose fitness only once every four years, and their reproductive output during years of little or no herbivory will more than compensate for the fitness lost to herbivores on the heavy herbivory years . The above explanation would hold only to the extent that there are no carry - over effects from one year to another . More recently, mccall cited bierzychudek and eckhart and delph in support of the claim that the reproductive output of females is more limited by resources than that of males . However, while there may be ample evidence that reproductive allocation is generally greater for the female function, it is possible that, upon the separation of sexes, physiological mechanisms involved in resource acquisition and allocation evolve in such way as to minimize the differences in reproductive effort between sexes . For example, in ilex glabra, sexes do not differ in total reproductive biomass produced in a growing season because the greater unitary investment in pistillate flowers and fruit development is negated by the sevenfold greater flower production in males . Given the importance of the tenet of greater female reproductive allocation for the expectation of male - biased herbivory, all studies of sex - biased herbivory should test for intersexual differences in reproductive allocation or provide a reference to an empirical study that demonstrates such differences for the species in question . In the measurement of reproductive allocation, particular attention must be paid to obtaining reliable estimates of male reproductive output (pollen production), which presents its own logistical difficulties . In addition, resource expenditure in pollinator attraction needs to be considered, as this is another expenditure related to reproduction that may differ between sexes . In essence, the presumed chain of evolutionary events that lead to male - biased herbivory in dioecious plants stems from the reallocation of those resources freed upon the loss of a sexual function in unisexual mutants towards defence . In most studies however, defence may also occur through tolerance [22, 93]. In comparison to resistance traits, tolerance traits have been more elusive . The capacity to store and mobilize carbohydrates, the presence of meristems and the capacity to activate them in response to damage growth rate has also been proposed to influence tolerance, although it is controversial whether high or low growth rates favour tolerance [9498]. A recent model shows that plants with low growth rates are more tolerant to herbivore damage . This model also shows that plants that can change their growth rate positively in response to damage will tolerate damage better than those with a different response in growth rate . The activation of meristems and mobilization of resources in response to the loss of tissue are two well - documented responses [100, 101] that could contribute to increased plant growth rates in response to damage . Thus, according to this model, if females grow more slowly than males because of their greater allocation to reproduction, then females should be more tolerant to herbivory than males . Whether this prediction necessarily implies that females should be less resistant than males is not clear at this point since there is increasing evidence that these two modes of defence are not necessarily mutually exclusive [99, 103, 104]. There is one other possibility that has not been emphasized enough in the proposed models of the evolution of defence in dioecious species: while one possible consequence of a greater allocation of resources to reproduction in females is reduced allocation to growth, it is also possible that the main reduction in allocation is to defence . In this case, there would be no detectable detriment to growth . Consequently, female plants would suffer more damage (if they are less resistant; figure 1, path c) or greater fitness losses (if they are less tolerant) compared to males . Greater damage in females could lead to fewer, more spaced reproductive events or greater interannual variability in reproductive output either directly through a decrease in the availability of resources for reproduction or indirectly through a decrease in pollinator visitation rates due to a lack of resources needed for floral display or nectar production [75, 105108]. Fewer resources available for reproduction could also pose a selective pressure to become choosier about their mates, which may lead to increased fruit or seed abortion [75, 109, 110]. Evolutionary changes in the rate of resource acquisition in female individuals may occur through increased photosynthetic rates, canopy area, rates of mineral nutrient uptake, as well as greater branching of roots, and enhancement of mycorrhizal associations . A greater rate of resource acquisition in females would decrease the relative differences in costs of reproduction between sexes: the sex with the greater resource demand for reproduction would have an increased capacity to garner resources . Under such scenario, the life - time cost of reproduction at the individual level would be equal between sexes, thus eliminating the source of inequalities in the patterns of allocation between males and females . In summary, nothing dictates that there is only one evolutionary pathway regarding changes in the patterns of resource allocation among reproduction, growth, and defence following the evolution of unisexuality (figure 1). Alternatively, a stage in which female individuals have heavier damage levels because of resource limitation for resistance may be a transient evolutionary stage prior to the invasion of mutants whose greater defence levels are attained at the expense of growth . In this case, we should observe female - biased herbivory in younger dioecious lineages and male - biased herbivory in those lineages in which there has been enough time for selection to reshape the patterns of resource allocation to reproduction, defence, and growth . We should be able to test this by means of relative dating of lineages with male- or female - biased herbivory . Similarly, as long as there has not been selection on prereproductive growth rates following the evolution of unisexuality, we should not see differences in growth rates or other physiological vegetative traits between males and females before their first reproductive event . It is difficult to test this prediction without reliable morphological or genetic markers that allow juveniles to be sexed so that their performance can be compared on the basis of sex . Some sex - linked markers may be, effectively, sex - related traits expressed before the onset of reproduction . Whether the presence of these markers implies the existence of sex chromosomes is still an area in need of further investigation [84, 85, 111113]. In short, without fitness gain curves for each sex, it is difficult to predict accurately which sex should evolve greater resistance against herbivores and whether we should expect or not male - biased herbivory in dioecious species . In fact, we need fitness surfaces in order to include the effects of reductions in leaf area caused by herbivory . Moreover, the fitness surfaces should account for the short- and long - term responses of plants in terms of changes in photosynthetic rates, reallocation of resources to shoot or root, activation of meristems, and delays in phenology or shortening of life span brought about by herbivore damage [95, 99]. It has not escaped our attention that the evolution of defence in gynodioecious species can be approached from a similar perspective to the one presented above for dioecious species . It is important to consider that some of gynodioecious species may be in evolutionary transition to dioecy while others are not [112, 114]. Another important difference with dioecious species is that, in gynodioecious plants, the morph that performs the male function the hermaphrodites may have a greater cost of reproduction because of the expenditure of resources on two sexual functions . Does this mean that hermaphrodites would be less resistant (figure 1, path c) or grow more slowly and evolve greater resistance (figure 1, path b)? Clearly, making predictions with respect to gender dimorphism in defensive traits for bisexual conditions along the gradation from monoecy to dioecy is not straight forward . The collection of studies cited in the reviews of herbivory in dioecious species [72, 73] has, as a group, important shortcomings that weaken the conclusion of male - biased herbivory as a generality in dioecious species . The main shortcomings are (1) the taxonomic bias of the sample of species studied; and (2) failure to test for or provide references of empirical studies of intersexual differences in (a) resistance traits the purported cause of the intersexual differences in herbivory levels; (b) growth rates the purported cause of the intersexual differences in resistance to herbivore attack; and (c) reproductive effort the purported cause of the aforementioned intersexual differences in growth rates . These deficiencies had been pointed out earlier [72, 89], but judging by statements included in the introduction or discussion of many of the papers published after the 1999 review, such caveats have not been considered to their full extent, and many authors take for granted either the generality of male - biased herbivory in dioecious species or its expectation without reference to any theoretical context . Cornelissen and stiling's meta - analysis of sex - biased herbivory includes 30 species, 28 of which are angiosperms . Focusing only on angiosperms, 13 of the 28 species were not considered previously in gren et al . These 30 species represent a total of 20 genera, 18 families, and 10 orders . Nine of those species belong to the same genus: salix . Adding to those populus tremula, the species in the salicaceae represent one - third of all species considered for the meta - analysis . Such distribution contrasts greatly with the taxonomic distribution of dioecy in 14,620 species, 959 genera, 157 families, and 36 orders . Of the four dioecious genera (consisting of solely dioecious species) with most species (400), pandanus, diospyros, and litsea, with 700, 500 +, and 400 species, respectively (; s. renner, university of munich, unpublished data) have not been studied for sex - biased herbivory yet . Clearly, we need to direct our research efforts to the most understudied orders and families if we want to arrive at generalizations regarding the biology of dioecious species, and particularly the influence of herbivores in their ecology and evolution . In addition to this taxonomic bias, a critical reexamination of that list of species casts serious doubt on the conclusion that male - biased herbivory is a rule in dioecious species: only 13 of those species were reported invariably to have male - biased herbivory . This list includes three salix, two freycinetia, and two species for which evidence of male - biased herbivory has not been published: hippophae rhamnoides and rumex acetosa . (in fact, male - biased herbivory in myrica gale not included in these 13 species is also anecdotal .) Greater herbivore damage on females is reported for four species, while the rest show either no intersexual differences (16), or variation in the result, depending on different factors (species of herbivore, kind of herbivore, tissue damaged, time of year, phenological or ontogenetic stage, etc . ). Moreover, it is possible that the results for those 13 species would show variation with either population or site, had these factors been studied . Perhaps the most serious problem with several studies of herbivory and dioecy has been the failure to make the connection between sex - biased herbivore damage and intersexual differences in growth rate, precisely because the latter is the purported cause of the former . Of the 30 species of angiosperms in table 2, either growth rate or a surrogate variable for growth (e.g., shoot length) was measured only in 21 species . Males grew faster in six species, females in two, no difference between sexes was detected in six species, and three species showed variable results . It must be noted that the same number of species shows no difference between genders as those that show greater growth rate in males . Considering solely the 13 species that invariably showed male - biased herbivory, only two show a greater growth rate in males: acer negundo and hippophae rhamnoides . However, as the evidence for male - biased herbivory in h. rhamnoides is anecdotal, we are left with only one species for which growth rate was measured in the same study as herbivore damage: acer negundo . Only 12 of the 30 species listed were assessed for intersexual differences in reproductive allocation in terms of reproductive effort (the proportion of biomass or other currency devoted to reproductive structures relative to the total biomass or expenditure in the selected currency of an individual). Reproductive effort was greater in females of 10 species and in males for the other two species . In some species, reproductive effort was measured during flowering, but allocation to fruit production was not considered (e.g., silene dioica). In those cases, we are left with an incomplete picture of reproductive allocation, and we can only join the authors in speculating whether species of the same genus have similar patterns of reproductive allocation . The only species that have been assessed for foliar damage, growth rate, and reproductive allocation in the same study are c. tepejilote, b. halimifolia, i. glabra, n. psychotrioides, and r. alpinus (table 2). These studies clearly made the chain of causal connections from sex bias in reproductive allocation all the way to sex bias in some resistance traits (except for c. tepejilote and i. glabra), and, as a consequence of the latter, sex bias in levels of damage . The study of r. alpinus went even further, comparing these attributes between pre- and postreproductive plants, and thus emphasizing that the root of the differences in growth rates, resistance traits, and leaf damage is in the patterns of reproductive resource expenditures [63, 116]. In some species, reproductive allocation, growth rate, and/or resistance however, even with these studies, the number of species for which we have a more complete picture of the causal links amongst these attributes remains low: nine more species (c. alternans, b. dracunculifolia . A. canescens, r. acetosella, s. caprea, s. cinerea, s. lasiolepis, s. sericeae, and h. rhamnoides [? ]; table 2) now have published data for damage and growth rate, bringing the number of species in this situation to 15 . Two more species, for a total of three, now have data on damage, growth rate, and reproductive allocation (c. alternans, s. dioica, and n. psychotrioides). Two more species now have data on reproductive allocation, growth rate, and resistance apart from herbivore damage, for a total of four species with all four variables measured (a. triphyllum, b. halimifolia, l. benzoin, and r. alpinus). In summary, the majority of studies on the topic of sex - biased herbivory have neglected the purported causal connections between bias in reproductive allocation, differential growth rate, resistance, and herbivore damage . Also, some authors seemed to confuse theoretical expectations with empirical evidence of greater female reproductive allocation: while lloyd and webb argue convincingly for the expectation of greater reproductive effort in females, they provided empirical evidence only for rumex acetosella, citing putwain and harper 1972 . Therefore, lloyd and webb's excellent paper cannot be cited as solid empirical evidence of greater reproductive effort in females . Lastly, anecdotal evidence should be taken with great caution and always flagged as such until data are published (see entries marked? In table 2). Using the search terms herbiv * and dioec * for entries between january 1998 and may 2012 on the web of science, we found nine studies encompassing 14 species that were not included in either of the previous reviews of the topic . Of these, only the study on the three species of chamaedorea palms measured reproductive allocation, growth rate, resistance, and herbivore damage (table 3; n.b . : one of these species had been studied before: c. alternans = c. tepejilote). Only one other study measured damage and reproductive allocation (sclerocarya birrea, table 3). Similarly, growth rate was assessed in only one other species (salix arctica). The taxonomic breadth of the studies of herbivory in dioecious species increased only by one family (in an unplaced order of the euasterids i). The general lack of consistency in the level of detail and the variables that have been measured in all these studies could be addressed if researchers interested in this topic followed a minimally standardized protocol . New studies must clearly allude to the theoretical framework from which the prediction of sex - biased herbivory levels (resistance) stems resource allocation theory, in particular, sex allocation . The claim that male - biased herbivory is expected because it has been reported as a pattern, whether implicit or explicit, lacks heuristic value because it does not address the causes of such pattern . Moreover, a plethora of factors may modify the expected pattern, as shown above . Clearly, we need to increase the taxonomic breadth of the studies of herbivory in dioecious species . We could direct our attention to those families with the greatest number of dioecious species or those with the greatest proportion of dioecious species . The first alternative will miss families with low species richness that may have a high proportion of dioecious species . The second method will miss families with high species richness but low proportion of dioecious species . One possible compromise is to focus our studies on the families with the greatest number of dioecious species among those with a large proportion of dioecious species, for instance, 50% or more (table 4). So far, we have studied only 2% of the dioecious species in the most studied family (salicaceae). If we took that as a target, we would have to study about 155 species for the 30 most dioecious species - rich families of angiosperms . However, by this method we would include only one species per family for many families, thus failing to achieve adequate representation of those families . In addition, we must consider that the conditions that determine sex - biased herbivory can change with habitat, and therefore some species may need to be studied in several habitats . In addition to the taxonomic bias, there is a preponderance of studies of woody plants . While this is understandable because most dioecious species are woody, we should strive for representation of the herbaceous component . With increased research on herbaceous dioecious species, we can address the influence of life history traits on the evolution of dioecy and defence . Lastly, we propose that all studies aimed at assessing whether herbivory levels differ between sexes and whether these differences are a consequence of differential growth rates (in turn resulting from differential allocation to reproduction) should conduct, at least, the following measurements and observations: (1) levels of herbivory, measured as precisely as possible (preferably for more than one growing season in perennials); (2) species of herbivores responsible for most of the damage; (3) growth rates, measured either as rgr for whole individuals or from increments in branch length or leaf production; (4) reproductive allocation, measured both as the number of reproductive structures (flowers and pollen production for males, flowers, and fruits for females), and also as reproductive effort (the proportion of individual or shoot biomass allocated to reproduction, and when possible n and p allocation to reproductive structures); and finally (5) the most important resistance characters that could be influencing the levels of herbivory and measure them quantitatively . In addition, these studies could add an experimental component in which plants are damaged at least at the highest rate seen in the surveys of natural damage, so as to measure tolerance to herbivory as well as resistance [75, 102]. Ideally, damage should be performed by placing natural herbivores on the plants because mechanical damage does not necessarily elicit the same physiological responses as herbivore damage . Also, these studies should consider that resistance and tolerance may vary both with ontogeny and with respect to other reproductive phenology because the acquisition and expenditure of resources vary at different stages of development and life history [119121]. We must reiterate that other authors have emphasized the need to address several of the points outlined above . It is our hope that future studies take these recommendations seriously so that we have to assume and speculate less, and we have empirical data to further our understanding of the evolution of defence in dioecious species . The study of the evolution of sex - biased herbivory is hampered by the notion that male - biased herbivory in dioecious species is a rule . We have presented other possible evolutionary outcomes with regards to sex - biased herbivory in the transition from hermaphroditic populations to dioecious ones . We have also discussed how these different outcomes can be predicted under different theoretical assumptions . Therefore, future studies of herbivory in dioecious species should be based on a clear theoretical framework . In particular, we urge that all new studies of herbivory in dioecious species include assessments of reproductive allocation, growth rates, and resistance traits deemed to differ between sexes and, therefore, determine sex - biased herbivory . In addition, tolerance should also be considered as a potentially important defence mode that can vary between sexes . In this manner the advancement of our knowledge about sex - related defence in plants should help us gain a better understanding of the evolution of sex - related traits in general.
Hepatocellular carcinoma (hcc) is the fifth most common cause of cancer, and its incidence is increasing worldwide because of the dissemination of hepatitis b and c virus infection15 . Patients with cirrhosis are at the highest risk of developing hcc and should be monitored every 6 months to diagnose the tumour at an asymptomatic stage68 . In most solid malignancies, most patients with hcc, however, have two diseases liver cirrhosis and hcc and complex interactions between the two have major implications for prognosis and treatment choice9 . The system that links staging with treatment modalities in hcc is the barcelona clinic liver cancer (bclc) staging system4,5,10 (table 1). It includes variables related to tumour stage, liver functional status (child pugh classification, table 2), physical status and cancer related symptoms (world health organization performance status, table 3)11,12 . Table 1bclc staging system in patients diagnosed with hcc 4,5,10very early stageps 0, child b, portal neoplastic invasion, nodal metastases, distant metastasesterminal stageps> 2, child table 2child pugh classification of severity of liver disease11parameterpoints assigned123ascitesabsentmild / moderatesevere / refractorybilirubin (mg / dl)223>3albumin (g / dl)>3.52.83.5<2.8prothrombin time seconds over control<446>6 inr<1.71.72.3>2.3encephalopathynonemildseverea total score of 56 is considered grade a (well - compensated disease); 79 is grade b (significant functional compromise); and 1015 is grade c (decompensated disease). Table 3world health organization performance status grades 12stage 0fully active, normal life, no symptomsstage 1minor symptoms, able to do light activitystage 2capable of self - care but unable to carry out work activities; up for more than 50% waking hoursstage 3limited self care capacity; confined to bed or chair> 50% waking hoursstage 4completely disabled; confined to bed or chair bclc staging system in patients diagnosed with hcc 4,5,10 hcc, hepatocellular carcinoma; ps, performance status . Child pugh classification of severity of liver disease11 a total score of 56 is considered grade a (well - compensated disease); 79 is grade b (significant functional compromise); and 1015 is grade c (decompensated disease). World health organization performance status grades 12 in the bclc system, early - stage hcc includes patients with who performance status of 0, preserved liver function (child pugh class a or b), and solitary tumour or up to 3 nodules each smaller than 3 cm in size, in the absence of macroscopic vascular invasion and extrahepatic spread . If the patient has child pugh class a cirrhosis and a solitary tumour smaller than 2 cm in size, the stage may be defined as very early . Patients with multinodular hcc with neither vascular invasion nor extrahepatic spread are classified as intermediate - stage according to the bclc staging system, provided that they have a performance status of 0 and child the terminal stage includes patients who have either severe hepatic decompensation (child pugh class c) or performance status greater than 2 (table 1). Patients with early - stage hcc can benefit from curative therapies, including surgical resection, liver transplantation and percutaneous ablation, and have the possibility of long term cure, with 5-year survival figures ranging 5075%5 . Resection is the treatment of choice for hcc in non - cirrhotic patients, who account for about 5% of the cases in western countries . However, in patients with cirrhosis, candidates for resection have to be carefully selected to reduce the risk of postoperative liver failure . It has been shown that a normal bilirubin concentration and the absence of clinically significant portal hypertension are the best predictors of excellent outcomes after surgery13 . In experienced hands, such patients less than 5% of cirrhotic patients with hcc have treatment - related mortality of less than 13% and may achieve a 5-year survival higher than 70%1315 . In contrast, survival drops to less than 50% at 5 years in patients with significant portal hypertension, and to less than 30% at 5 years in those with both adverse factors (portal hypertension and elevated bilirubin)13 . Liver transplantation is the only option that provides cure of both the tumour and the underlying chronic liver disease . It is recognized as the best treatment for patients with solitary hcc smaller than 5 cm in the setting of decompensated cirrhosis and for those with early multifocal disease (up to 3 lesions, none larger than 3 cm)1 . The reported outcomes of patients who actually underwent transplantation are better than those of patients submitted to resection, especially if the substantially lower rates of tumour recurrence less than 1020% versus more than 70% at 5 years are considered13,16 . In fact, because of the lack of sufficient liver donation, there is always a waiting period between listing and transplantation, during which the tumour may grow and develop contraindications to transplantation (vascular invasion, extrahepatic spread). The rate of dropouts may be as high as 25% if the waiting list is longer than 12 months19 . This difficulty may be in part overcome by living donation, that, however, has inherent limitations and requires a highly skilled surgical team16 . Image - guided percutaneous ablation is currently accepted as the best therapeutic choice for non - surgical patients with early - stage hcc4,5 . Over the past two decades, several methods for chemical ablation or thermal tumour destruction have been developed and clinically tested . These include the injection of ethanol or acetic acid and the administration of localized heating (radiofrequency, microwave, laser ablation) or freezing (cryoablation). The seminal technique used for local ablation of hcc is percutaneous ethanol injection (pei). Ethanol induces coagulation necrosis of the lesion as a result of cellular dehydration, protein denaturation, and chemical occlusion of small tumour vessels . Pei is a well - established technique for the treatment of nodular - type hcc . Hcc nodules have a soft consistency and are surrounded by a firm cirrhotic liver . Consequently, injected ethanol diffuses within them easily and selectively, leading to complete necrosis of about 70% of small lesions20 . Although there have not been any rct comparing pei and best supportive care or pei and surgical resection, several retrospective studies have provided indirect evidence that pei improves the natural history of hcc: the long - term outcomes of patients with small tumours who were treated with pei were similar to those reported in surgical series, with 5-year survival rates ranging 4160% in child a patients2025 . Of importance, two cohort studies and one retrospective case control study comparing surgical resection and pei failed to identify any difference in survival, despite the fact that the patients in pei groups had poorer liver function2628 . Pre - treatment ct obtained in the arterial (a) and the delayed phase (b) shows the lesion as a small hypervascular nodule (a). Injection of an ultrasound contrast agent confirms arterial - phase enhancing tumour (arrow, c). B - mode low - mechanical index image is shown in the left side of the image; the contrast - specific mode during the arterial phase is shown in the right side . The tumour is treated with rf ablation under us guidance . At the end of the procedure, a large hyperechoic cloud covering the tumour as well as a cuff of surrounding liver parenchyma is seen on us (d). Contrast - enhanced ultrasound study performed at the end of the procedure shows the ablation zone as an unenhanced area completely covering the tumour . Peri - ablation enhancement is also seen, representing reactive hyperaemia (e). On ct images obtained in the arterial (f) and the portal venous phase (g) 1 month after treatment, pre - treatment ct obtained in the arterial (a) and the delayed phase (b) shows the lesion as a small hypervascular nodule (a). Injection of an ultrasound contrast agent confirms arterial - phase enhancing tumour (arrow, c). B - mode low - mechanical index image is shown in the left side of the image; the contrast - specific mode during the arterial phase is shown in the right side . The tumour is treated with rf ablation under us guidance . At the end of the procedure, a large hyperechoic cloud covering the tumour as well as a cuff of surrounding liver parenchyma contrast - enhanced ultrasound study performed at the end of the procedure shows the ablation zone as an unenhanced area completely covering the tumour . On ct images obtained in the arterial (f) and the portal venous phase (g) 1 month after treatment, the tumour is replaced by a non - enhancing ablation zone . The major limitation of pei is the high local recurrence rate, which may reach 33% in lesions smaller than 3 cm and 43% in lesions exceeding 3 cm29,30 . The injected ethanol does not always accomplish complete tumour necrosis because of its inhomogeneous distribution within the lesion especially in the presence of intratumoural septa and the limited effect on extracapsular cancerous spread . Moreover, pei is unable to create a safety margin of ablation in the liver parenchyma surrounding the nodule, and therefore may not destroy tiny satellite lesions that even in small tumours may be located in close proximity to the main nodule . Application of localized heating or freezing enables in situ destruction of malignant liver tumours preserving normal liver parenchyma . The thermal ablative therapies involved in clinical practice can be classified as either hepatic hyperthermic treatments including radiofrequency (rf) ablation, microwave ablation, and laser ablation or hepatic cryotherapy . Hepatic hyperthermic treatments are mostly performed via a percutaneous approach; until recently, an open or laparoscopic approach was widely adopted for hepatic cryotherapy . The thermal damage caused by heating is dependent on both the tissue temperature achieved and the duration of heating . Heating of tissue at 5055c for 46 min produces irreversible cellular damage . At temperatures between 60c and 100c near immediate coagulation of tissue is induced, with irreversible damage to mitochondrial and cytosolic enzymes of the cells . At more than 100110c, tissue vaporizes and carbonizes31 . For adequate destruction of tumour tissue, the entire target volume different physical mechanisms are involved in the hepatic hyperthermic treatments in order to generate a lethal temperature . A common important factor that affects the success of thermal ablation is the ability to ablate all viable tumour tissue and possibly an adequate tumour - free margin . Ideally, a 360, 0.51-cm - thick ablative margin should be produced around the tumour31 . This cuff would ensure that microscopic invasions around the periphery of a tumour have been eradicated . Thus, the target diameter of an ablation, or of overlapping ablations, must be larger than the diameter of the tumour undergoing treatment32 . Thermal ablation is usually performed under intravenous sedation with standard cardiac, pressure, and oxygen monitoring . Targeting of the lesion can be performed with ultrasound, computed tomography (ct), or magnetic resonance (mr) imaging . The guidance system is chosen largely on the basis of operator preference and local availability of dedicated equipment such as ct fluoroscopy or open mr systems . Real - time ultrasound / ct (or ultrasound / mr imaging) fusion imaging substantially improves the ability to guide and monitor liver tumour ablation procedures . Current virtual navigation systems can help to define the extent of the liver tumour burden, plan and simulate the insertion of the needle, and predict the amount of the induced necrosis . During the procedure, important aspects to be monitored include how well the tumour is being covered and whether any adjacent normal structures are being affected at the same time . While the transient hyperechoic zone that is seen at ultrasound within and surrounding a tumour during and immediately after rf ablation can be used only as a rough guide to the extent of tumour destruction, mr is currently the only imaging modality with validated techniques for real - time temperature monitoring . At the end of the procedure, the needle track can be ablated in most systems, aimed at preventing any tumour cell dissemination . Contrast - enhanced ultrasound performed at the end of the procedure may allow an initial evaluation of the effect of the treatment . However, contrast - enhanced ct and mr imaging are recognized as the standard modalities to assess treatment outcome . Ct and mr images obtained after treatment show successful ablation as a non - enhancing area with or without a peripheral enhancing rim . The enhancing rim that may be observed along the periphery of the ablation zone appears a relatively concentric, symmetric area, with smooth inner margins . This is a transient finding that represents a benign physiologic response to thermal injury (initially, reactive hyperaemia; subsequently, fibrosis and giant cell reaction). Benign peri - ablational enhancement needs to be differentiated from irregular peripheral enhancement due to residual tumour that occurs at the treatment margin . In contrast to benign peri - ablational enhancement, residual unablated tumour often grows in scattered, nodular, or eccentric patterns33 . Later follow - up imaging studies should be aimed at detecting the recurrence of the treated lesion (i.e. Local tumour progression), the development of new hepatic lesions, or the emergence of extrahepatic disease . The goal of rf ablation is to induce thermal injury to the tissue through electromagnetic energy deposition . The patient is part of a closed - loop circuit, that includes an rf generator, an electrode needle, and a large dispersive electrode (ground pads). Because of the relatively high electrical resistance of tissue in comparison with the metal electrodes, there is marked agitation of the ions present in the target tissue that surrounds the electrode, since the tissue ions attempt to follow the changes in direction of alternating electric current . The discrepancy between the small surface area of the needle electrode and the large area of the ground pads causes the generated heat to be focused and concentrated around the needle electrode . Several electrode types are available for clinical rf ablation, including internally cooled electrodes and multitined expandable electrodes with or without perfusion33 . Rf ablation has been the most widely assessed alternative to pei for local ablation of hcc34 . Histologic data from explanted liver specimens in patients who underwent rf ablation showed that tumour size and the presence of large (3 mm or more) abutting vessels significantly affect the local treatment effect . Complete tumour necrosis was pathologically shown in 83% of tumours less than 3 cm and 88% of tumours in a non - perivascular location35 . Three rcts compared rf ablation versus pei for the treatment of early - stage hcc3638 . The first trial, performed in european centres, failed to show a statistically significant difference in overall survival between patients who received rf ablation and those treated with pei36 . However, survival advantages were identified in a subgroup analysis of a trial coming from taiwan37 and in a japanese study, although in the latter the survival benefit was not confirmed in the subgroup analysis of patients with solitary tumours38 . All three investigations showed that rf ablation had a higher local anticancer effect than pei, leading to better local control of the disease . Therefore, rf ablation appears as the preferred percutaneous treatment for patients with early - stage hcc on the basis of more consistent local tumour control . Recently, the long - term survival outcomes of rf ablation - treated patients were reported (table 4). In the first published report, 206 patients with early - stage hcc who were not candidates for resection or transplantation were enrolled in a prospective, intention - to - treat clinical trial39 . Rf ablation was considered as the first - line non - surgical treatment and was actually performed in 187 (91%) of 206 patients . Nineteen (9%) of 206 patients had to be excluded from rf treatment because of the unfavourable location of the tumour . In patients who underwent rf ablation, survival depended on the severity of the underlying cirrhosis and the tumour multiplicity . Patients in child class a with solitary hcc had a 5-year survival rate of 61% . Three other studies confirmed that the survival of naive patients (i.e. Patients who received radiofrequency ablation as primary treatment) with well - compensated cirrhosis bearing early - stage hcc ranged from 43% to 64%4042 (table 4). Of interest, in a randomized trial of rf ablation versus surgical resection in patients with solitary hcc less than 5 cm in diameter, no differences in overall survival rates and cumulative recurrence - free survival rates were observed43 . Table 4studies reporting long - term survival outcomes of patients with early - stage hepatocellular carcinoma who underwent percutaneous radiofrequency ablationauthor and yearno . Patientssurvival rates (%) 1 year3 years5 yearslencioni et al ., 200539child a, 1 hcc <5 cm or 3 <3 cm14410076511 hcc <5 cm1161008961child b, 1 hcc <5 cm or 3 <3 cm43894631tateishi et al ., 200540naive patients319957854non - naive patients345926238cabassa et al ., 20064159946543choi et al ., 200742child a, 1 hcc <5 cm or 3 <3 cm359na7864child b, 1 hcc <5 cm or 3 <3 cm160na4938hcc, hepatocellular carcinoma.patients who received radiofrequency ablation as primary treatment.patients who received radiofrequency ablation for recurrent tumour after previous treatment including resection, ethanol injection, microwave ablation, and transarterial embolization . Studies reporting long - term survival outcomes of patients with early - stage hepatocellular carcinoma who underwent percutaneous radiofrequency ablation hcc, hepatocellular carcinoma . Patients who received radiofrequency ablation as primary treatment . Patients who received radiofrequency ablation for recurrent tumour after previous treatment including resection, ethanol injection, microwave ablation, and transarterial embolization . Recently, three separate multicentre surveys have reported mortality rates ranging from 0.1% to 0.5%, major complication rates ranging from 2.2% to 3.1%, and minor complication rates ranging from 5% to 8.9%44 . The most common causes of death were sepsis, hepatic failure, colon perforation, and portal vein thrombosis, while the most common complications were intraperitoneal bleeding, hepatic abscess, bile duct injury, hepatic decompensation, and grounding pad burns4547 . An uncommon late complication of rf ablation is tumour seeding along the needle track . In patients with hcc, tumour seeding occurred in 8 (0.5%) of 1610 cases in a multicentre survey45 and in 1 (0.5%) of 187 cases in a single - institution series39 . Lesions with subcapsular location and an invasive tumoural pattern, as shown by a poor degree of differentiation, seem to be at higher risk for such a complication48 . While these data indicate that rf ablation is a relatively safe procedure, a careful assessment of the risks and benefits associated with the treatment has to be made in each individual patient by a multidisciplinary team . Despite the many published reports, some questions concerning image - guided rf ablation in hcc treatment are still unanswered . Some authors have reported that rf ablation may be a safe and effective bridge to liver transplantation4952 . However, randomized studies would be needed to determine the advantages and disadvantages of rf ablation with respect to transarterial chemoembolization (tace) for hcc patients awaiting transplantation . Recent studies have reported encouraging initial results in the treatment of intermediate - size hcc lesions with a combination of rf ablation and balloon catheter occlusion of the tumour arterial supply or prior tace5357 . However, further clinical trials are warranted to determine the survival benefit associated with this approach . Microwave ablation is the term used for all electromagnetic methods of inducing tumour destruction by using devices with frequencies greater than or equal to 900 khz . The passage of microwaves into cells or other materials containing water results in the rotation of individual molecules . This rapid molecular rotation generates and uniformly distributes heat, which is instantaneous and continuous until the radiation is stopped . Microwave irradiation creates an ablation area around the needle in a column or round shape, depending on the type of needle used and the generating power58 . The local effect of treatment in hcc was assessed by examining the histological changes of the tumour after microwave ablation59,60 . Coagulative necrosis with faded nuclei and eosinophilic cytoplasm were the predominant findings in the ablated areas . There were also areas in which the tumours maintained their native morphological features as if the area was fixed, but their cellular activity was destroyed as demonstrated by succinic dehydrogenase stain . One study compared microwave ablation and pei in a retrospective evaluation of 90 patients with small hcc61 . The overall 5-year survival rates for patients with well - differentiated hcc treated with microwave ablation and pei were not significantly different . However, among the patients with moderately or poorly differentiated hcc, overall survival with microwave ablation was significantly better than with pei . In a large series including 234 patients, the 3- and 5-year survival rates were 73% and 57%, respectively62 . At a multivariate analysis, tumour size, number of nodules, and child pugh classification had a significant effect on survival63 . Only one randomized trial compared the effectiveness of microwave ablation with that of rf ablation64 . Seventy - two patients with 94 hcc nodules were randomly assigned to rf ablation and microwave ablation groups . Unfortunately, in this study the data were analyzed with respect to lesions and not to patients . Although no statistically significant differences were observed with respect to the efficacy of the two procedures, a tendency favouring rf ablation was recognized with respect to local recurrences and complications rates . The term laser ablation should be used for ablation with light energy applied via fibres directly inserted into the tissue . In addition, different types of laser fibres, modified tips, and single or multiple laser applicators can be used . From a single, bare 400-m laser fibre, a spherical volume of coagulative necrosis up to 2 cm in diameter can be produced . The first consists of firing multiple bare fibres arrayed at 2-cm spacing throughout a target lesion; the second uses cooled - tip diffuser fibres that can deposit up to 30 w over a large surface area, thus diminishing local overheating65 . To date, few data are available concerning the clinical efficacy of laser ablation . No randomized trials to compare laser ablation with any other treatment have been published thus far . In one study including 74 patients with early - stage hcc, overall survival rates were 68% at 3 years and 15% at 5 years, respectively66 . Laser ablation appears to be relatively safe, with a major complication rate less than 2%67 . The major drawback of current laser technology appears to be the small volume of ablation that can be created with a single - probe insertion . Insertion of multiple fibres is technically cumbersome and may not be feasible in lesions that are not conveniently located . The goal of rf ablation is to induce thermal injury to the tissue through electromagnetic energy deposition . The patient is part of a closed - loop circuit, that includes an rf generator, an electrode needle, and a large dispersive electrode (ground pads). Because of the relatively high electrical resistance of tissue in comparison with the metal electrodes, there is marked agitation of the ions present in the target tissue that surrounds the electrode, since the tissue ions attempt to follow the changes in direction of alternating electric current . The discrepancy between the small surface area of the needle electrode and the large area of the ground pads causes the generated heat to be focused and concentrated around the needle electrode . Several electrode types are available for clinical rf ablation, including internally cooled electrodes and multitined expandable electrodes with or without perfusion33 . Rf ablation has been the most widely assessed alternative to pei for local ablation of hcc34 . Histologic data from explanted liver specimens in patients who underwent rf ablation showed that tumour size and the presence of large (3 mm or more) abutting vessels significantly affect the local treatment effect . Complete tumour necrosis was pathologically shown in 83% of tumours less than 3 cm and 88% of tumours in a non - perivascular location35 . Three rcts compared rf ablation versus pei for the treatment of early - stage hcc3638 . The first trial, performed in european centres, failed to show a statistically significant difference in overall survival between patients who received rf ablation and those treated with pei36 . However, survival advantages were identified in a subgroup analysis of a trial coming from taiwan37 and in a japanese study, although in the latter the survival benefit was not confirmed in the subgroup analysis of patients with solitary tumours38 . All three investigations showed that rf ablation had a higher local anticancer effect than pei, leading to better local control of the disease . Therefore, rf ablation appears as the preferred percutaneous treatment for patients with early - stage hcc on the basis of more consistent local tumour control . Recently, the long - term survival outcomes of rf ablation - treated patients were reported (table 4). In the first published report, 206 patients with early - stage hcc who were not candidates for resection or transplantation were enrolled in a prospective, intention - to - treat clinical trial39 . Rf ablation was considered as the first - line non - surgical treatment and was actually performed in 187 (91%) of 206 patients . Nineteen (9%) of 206 patients had to be excluded from rf treatment because of the unfavourable location of the tumour . In patients who underwent rf ablation, survival depended on the severity of the underlying cirrhosis and the tumour multiplicity . Patients in child class a with solitary hcc had a 5-year survival rate of 61% . Three other studies confirmed that the survival of naive patients (i.e. Patients who received radiofrequency ablation as primary treatment) with well - compensated cirrhosis bearing early - stage hcc ranged from 43% to 64%4042 (table 4). Of interest, in a randomized trial of rf ablation versus surgical resection in patients with solitary hcc less than 5 cm in diameter, no differences in overall survival rates and cumulative recurrence - free survival rates were observed43 . Table 4studies reporting long - term survival outcomes of patients with early - stage hepatocellular carcinoma who underwent percutaneous radiofrequency ablationauthor and yearno ., 200539child a, 1 hcc <5 cm or 3 <3 cm14410076511 hcc <5 cm1161008961child b, 1 hcc <5 cm or 3 <3 cm43894631tateishi et al ., 200540naive patients319957854non - naive patients345926238cabassa et al ., 200742child a, 1 hcc <5 cm or 3 <3 cm359na7864child b, 1 hcc <5 cm or 3 <3 cm160na4938hcc, hepatocellular carcinoma.patients who received radiofrequency ablation as primary treatment.patients who received radiofrequency ablation for recurrent tumour after previous treatment including resection, ethanol injection, microwave ablation, and transarterial embolization . Studies reporting long - term survival outcomes of patients with early - stage hepatocellular carcinoma who underwent percutaneous radiofrequency ablation hcc, hepatocellular carcinoma . Patients who received radiofrequency ablation as primary treatment . Patients who received radiofrequency ablation for recurrent tumour after previous treatment including resection, ethanol injection, microwave ablation, and transarterial embolization . Recently, three separate multicentre surveys have reported mortality rates ranging from 0.1% to 0.5%, major complication rates ranging from 2.2% to 3.1%, and minor complication rates ranging from 5% to 8.9%44 . The most common causes of death were sepsis, hepatic failure, colon perforation, and portal vein thrombosis, while the most common complications were intraperitoneal bleeding, hepatic abscess, bile duct injury, hepatic decompensation, and grounding pad burns4547 . Minor complications and side effects were usually transient and self - limiting . An uncommon late complication of rf ablation is tumour seeding along the needle track . In patients with hcc, tumour seeding occurred in 8 (0.5%) of 1610 cases in a multicentre survey45 and in 1 (0.5%) of 187 cases in a single - institution series39 . Lesions with subcapsular location and an invasive tumoural pattern, as shown by a poor degree of differentiation, seem to be at higher risk for such a complication48 . While these data indicate that rf ablation is a relatively safe procedure, a careful assessment of the risks and benefits associated with the treatment has to be made in each individual patient by a multidisciplinary team . Despite the many published reports, some questions concerning image - guided rf ablation in hcc treatment are still unanswered . Some authors have reported that rf ablation may be a safe and effective bridge to liver transplantation4952 . However, randomized studies would be needed to determine the advantages and disadvantages of rf ablation with respect to transarterial chemoembolization (tace) for hcc patients awaiting transplantation . Recent studies have reported encouraging initial results in the treatment of intermediate - size hcc lesions with a combination of rf ablation and balloon catheter occlusion of the tumour arterial supply or prior tace5357 . However, further clinical trials are warranted to determine the survival benefit associated with this approach . Microwave ablation is the term used for all electromagnetic methods of inducing tumour destruction by using devices with frequencies greater than or equal to 900 khz . The passage of microwaves into cells or other materials containing water results in the rotation of individual molecules . This rapid molecular rotation generates and uniformly distributes heat, which is instantaneous and continuous until the radiation is stopped . Microwave irradiation creates an ablation area around the needle in a column or round shape, depending on the type of needle used and the generating power58 . The local effect of treatment in hcc was assessed by examining the histological changes of the tumour after microwave ablation59,60 . In one study, coagulative necrosis with faded nuclei and eosinophilic cytoplasm were the predominant findings in the ablated areas . There were also areas in which the tumours maintained their native morphological features as if the area was fixed, but their cellular activity was destroyed as demonstrated by succinic dehydrogenase stain . One study compared microwave ablation and pei in a retrospective evaluation of 90 patients with small hcc61 . The overall 5-year survival rates for patients with well - differentiated hcc treated with microwave ablation and pei were not significantly different . However, among the patients with moderately or poorly differentiated hcc, overall survival with microwave ablation was significantly better than with pei . In a large series including 234 patients, the 3- and 5-year survival rates were 73% and 57%, respectively62 . At a multivariate analysis, tumour size, number of nodules, and child pugh classification had a significant effect on survival63 . Only one randomized trial compared the effectiveness of microwave ablation with that of rf ablation64 . Seventy - two patients with 94 hcc nodules were randomly assigned to rf ablation and microwave ablation groups . Unfortunately, in this study the data were analyzed with respect to lesions and not to patients . Although no statistically significant differences were observed with respect to the efficacy of the two procedures, a tendency favouring rf ablation was recognized with respect to local recurrences and complications rates . The term laser ablation should be used for ablation with light energy applied via fibres directly inserted into the tissue . In addition, different types of laser fibres, modified tips, and single or multiple laser applicators can be used . From a single, bare 400-m laser fibre, a spherical volume of coagulative necrosis up to 2 cm in diameter can be produced . The first consists of firing multiple bare fibres arrayed at 2-cm spacing throughout a target lesion; the second uses cooled - tip diffuser fibres that can deposit up to 30 w over a large surface area, thus diminishing local overheating65 . To date, few data are available concerning the clinical efficacy of laser ablation . No randomized trials to compare laser ablation with any other treatment have been published thus far . In one study including 74 patients with early - stage hcc, overall survival rates were 68% at 3 years and 15% at 5 years, respectively66 . Laser ablation appears to be relatively safe, with a major complication rate less than 2%67 . The major drawback of current laser technology appears to be the small volume of ablation that can be created with a single - probe insertion . Insertion of multiple fibres is technically cumbersome and may not be feasible in lesions that are not conveniently located . Several image - guided ablation techniques have been developed to treat non - surgical patients with hcc . Percutaneous ablation is accepted as the best therapeutic choice for patients with early - stage hcc when resection or transplantation are precluded . On the basis of the current evidence, rf ablation seems to offer higher cumulative survival and recurrence - free survival rates compared with other image - guided treatments and is accepted as the primary ablative modality at most institutions . Further trials are needed to establish the clinical value of image - guided ablation in combination with intra - arterial treatments68.
Yogic visual concentration (trataka) is one of the six cleansing techniques mentioned in the ancient indian yogic text, hata yoga pradipika . Looking intently with an unwavering gaze at a small point until tears are shed is known as trataka (hata yoga pradipika, ch:2, v: 31). Hata yoga pradipika mentions that the practice of trataka eradicates all eye diseases, fatigue, and lethargy (hata yoga pradipika, ch:2, v: 32). Although trataka is known as a cleansing technique, the final stage of trataka induces a meditative mental state . When meditation is practiced over a period of time it improves perception, attention, and cognition . A large number of research studies have shown improvement in attentional task performance following meditation . The stroop color word test is a useful and reliable assessment tool used in psychology.4, 5 it was first described by john ridley stroop in 1935 . It measures selective attention, cognitive flexibility, and reaction time . A study used stroop color word test to examine the differences in various domains of attention between long - term concentrative meditators versus matched controls . Performance on the stroop test was significantly higher following long - term vihangam meditation practice suggesting an increase in selective attention, cognitive flexibility, and processing speed . Recently, a study was conducted to evaluate the immediate effect of trataka on critical flicker fusion (cff). Cff is defined as the frequency at which a flickering stimulus is perceived to be continuous . Thirty healthy volunteers were assessed in two sessions, i.e., a trataka and a control session . There was a significant increase in cff following trataka, suggesting changes at the cortical level in the processes that mediates fusion . However, no studies evaluating the immediate effect of trataka on cognitive performance, suggesting that there are changes, exist . Hence, the present study was designed to assess the immediate effect of trataka on cognitive performance using the stroop color word test . Thirty male volunteers with ages ranging from 18 years to 31 years old (22.57 3.65 years) were recruited for the study . They were all students of a yoga university in southern india . The predetermined conditions to exclude participants from the trial were chronic illness, visual deformities, and color blindness . The project was approved by the institution s ethics committee (swami vivekananda yoga anusandhana samsthana (a deemed university) bangalore). The study protocol was explained to the participants and their signed consent was obtained . The adult s version of the stroop color word test was used to assess the cognitive function of the participants . The first page tests how fast the participant can read the words, the second page tests how fast the participants can name the colors on the page, and on the third page the participants were asked to name the color of the ink the individual words were printed in, ignoring the word itself for each item . If the participants made a mistake, they were asked to stop and proceed after correcting the mistake . A stop - watch was used to record the time taken to complete the task . Each participant was assessed during two sessions (a trataka and a control session) on two separate days . Half of the participants practiced trataka on the 1 day and the control session was carried out on 2 day . Participants were alternately allocated to either schedule to prevent the order of the sessions influencing the outcome . The theoretical aspects of trataka were explained by a qualified yoga teacher on the 1 day . The first stage consists of eye exercises, which is a preparatory practice for trataka . The eye exercises include eyeball movements in horizontal, vertical, and diagonal directions and circular movements . Palming consists of putting slightly cupped palms over the eyes, so that the eyes perceive complete darkness . The participants were asked to fix their gaze on the flame of a candle for approximately 23 minutes, suppressing the urge to blink as far as possible . Finally, the participants were asked to defocus, and the practice ended with silence and prayer . The participants practiced eye exercises for 10 minutes, and then for the next 15 minutes, they sat quietly with their eyes closed without doing any concentration or meditation exercises . The stroop color word test yields three basic scores, namely: (1) raw word scores; (2) raw color scores; and (3) raw color word scores . The raw word score is the number of items completed on the word page, the raw color score is the number of items completed on the color page, and the raw color the pure interference score is calculated by subtracting the raw color score from the raw color statistical analysis was carried out using spss (ibm corporation, usa) (version 19.0). Since the same individuals were assessed in repeat sessions on separate days (i.e., trataka and control), repeated measures analysis of variance (rm anova) was used . Two - way rm anova was performed using two' within participants' factors, i.e., factor 1: sessions; trataka and control and factor 2: states; pre, and post . This was followed by a post - hoc analyses using bonferroni adjustment to compare pre with post values . The group mean and standard deviation for the scores obtained in the stroop color word test are presented in table 1 . Two - way rm anova showed a significant difference between sessions for: (1) word score f (1, 29) = 21.57, p <0.001; and (2) color score f (1, 29) = 9.65, p <0.01 . There was a significant difference between states for: (1) word score f (1, 29) = 163.42, p <0.001; (2) color score f (1, 29) = 195.30, p <0.001; and (3) color word score f (1, 29) = 435.24, p <0.001 . Also, there was a significant interaction between the session and state for (1 word score f (1, 29) = 55.69, p <0.001; (2) color score f (1, 29) = 29.61, p <0.001; and (3) color word score f (1, 29) = 54.90, p <0.001 . Post hoc analyses with bonferroni adjustment was performed and all comparisons were made using the respective pre states . There was a significant difference between the post session of trataka and of the control (p <0.001). There was a significant increase in the word score (p <0.001), color score (p <0.001), and color word score (p <0.001) after trataka compared to before trataka . And also, there was a significant increase in the word score (p <0.01), color score (p <0.001), and color word score (p <0.001) after the control session compared to before . In this study, stroop color word test was assessed before and after the practice of trataka and control sessions in 30 male volunteers . The stroop color word test is an index of executive functioning such as interference control, selective attention and cognitive flexibility, and response inhibition.9, 10, 11 trataka showed better performance on the stroop color word test compared to the control session . The stroop color word test consists of four basic scores, namely, the word score, the color score, the color word score, and the interference score . The word score is the number of items completed on the word page and it reflects basic reading speed . The word score significantly increased following trataka (15.63%) compared to the control session (4.42%), suggesting better reading speed after trataka . Naming of a color takes more time than naming a word because it requires a conscious effort to choose and say the name of the color . The color score is the number of items completed on the color page and it involves selective attention . The color score significantly increased after trataka (17.58%) compared to the control session (6.95%), suggesting better selective attention following trataka . Word score is the number of items completed on the color word page which involves naming the color of the ink the individual words are printed in, while ignoring the word that is printed for each item (i.e., the word red printed in green ink). Word page were significantly higher following trataka (26.05%) compared to the control session (10.68%), suggesting better selective attention, cognitive flexibility, and response inhibition after trataka . It is somewhat similar to a focused meditative state (dharana) which is described in the ancient yoga text, patanjali yoga sutras . A study compared performance on a cancellation task following four mental states namely, cancalata (random thinking), ekagrata (nonmeditative concentration), dharana (focused meditation), and dhyana (effortless meditation or meditative expansiveness). This suggests better selective attention, visual scanning, and concentration after the practice of dharana . In another study, an earlier study on trataka showed a significant increase in cff suggesting changes at the cortical level in the processes that mediates fusion . The findings of the present study are in line with this earlier study . In the current study, performance on the stroop color - word test is related to the prefrontal cortex . Hence, improved performance on the stroop color word test after trataka may be due to the increased activity at the prefrontal cortex . Though there was better performance after trataka compared to the control sessions there was a significant increase after the control session compared to presession scores . The control session consisted of 10 minute eye exercises, and for the following 15 minutes participants were asked to sit quietly with closed eyes without meditation . The participants may have practiced focused thinking or meditation during the control session and this may be the reason why there is an increase in the scores . However, this is just a speculation and no objective measurements were performed to confirm this . One of the main limitations of the study is that there is no guarantee that each participant did not practice meditation or any focused thinking during the control session . It would have been ideal to have a simultaneous assessment of physiological parameters to assess the level of autonomic arousal during the two sessions . Trataka showed better performance on the stroop color word test compared to the control session, suggesting increased selective attention, cognitive flexibility, and response inhibition following trataka . Further studies using other objective measurements would substantiate these findings and may help to understand the mechanisms involved.
Despite the pronounced improvement of oral and dental status, dental caries is still a common dental disease . The factor which almost affects the preventive dentistry performance is the knowledge and function of the medical group concerning this issue . An important aspect of the child's general care should involve the first dental visit . The quality of preventive health care and the future oral health is determined by the first dental visit . Recommendations for early dental visits for children suggest its completion by 12 months of age. [14] the medical office is considered an opportune site to reach large numbers of children who make a medical visit but not a dental visit . Physicians and their auxiliaries can assess risks for dental problems and counsel parents and their children about the prevention of these problems . They also can provide screening services for early detection of dental disease, provide advice about the need to seek dental care, and refer those in need . A key element of comprehensive care for children thus involves the coordination of services between medical and dental providers so that the appropriate health care professionals can provide appropriate services at the appropriate ages . Saliva is the most non - invasive and the most available body fluid containing inorganic and organic compounds that forms a mirror of the body's health offering a distinct advantage over serum in being cost effective . Saliva has been reliably used to detect hiv 1 and 2, and viral hepatitis a, b and c. it can also be used to monitor a variety of drugs including marijuana, cocaine and alcohol . This study conducted in the city of chennai and its suburbs is designed keeping in mind to gather the data on the level of knowledge of pediatric dentistry, importance of the first dental visit and referrals to a specialist for dental care . Even though similar studies have been recorded but this study uses information from baseline questionnaires, totaling 800, which are completed by various health care professionals . The list of pediatricians and family physicians were obtained from various private hospitals and associations in the year 2011 and the questionnaires were sent to them by mail as we felt that direct recording may affect the response . A comprehensive questionnaire were prepared based on studies done by yahya and solmaz, giuseppe et al ., georgia cruz et al . And prakash et al . The data was collected using a mailed self - administered questionnaire . The participants received a short questionnaire, a covering letter and an addressed, stamped return envelope . The items were evaluated through choosing one of the options completely, yes or no . The questionnaire was divided into the following four sections; individual details including: name, age, sex, medical backgroundthe approach of the practitioner towards pediatric dentistry and factors affecting it.the knowledge level of the practice of pediatric dentistry in relation to age at which care should be started, maternal considerations, preventive methods and various common treatment methods . Importance regarding early childhood caries (ecc) has been included in this part of the questionnaire along with the cariogenic potential of medicated syrups.training on oral health received by the practitioner were recorded and willingness to receive training . Individual details including: name, age, sex, medical background the approach of the practitioner towards pediatric dentistry and factors affecting it . The knowledge level of the practice of pediatric dentistry in relation to age at which care should be started, maternal considerations, preventive methods and various common treatment methods . Importance regarding early childhood caries (ecc) has been included in this part of the questionnaire along with the cariogenic potential of medicated syrups . Training on oral health received by the practitioner were recorded and willingness to receive training . A total of 800 questionnaires (400 - pediatricians, 400 - family physicians) were mailed and a total of 696 of questionnaires were returned duly completed out of which 371 were from pediatricians and 325 from general physicians and the results were tabulated in four sections and discussed . This survey was done with the need to emphasize on the fact that there is a need to improve the pediatrician / family physician and the pedodontist's relationship, as well as to study on the lacunae on the areas where the physicians need to improve the knowledge of oral health and to recognize the importance of pediatric dentistry . The approach of the practitioners [table 1] showed that more than 80% of pediatricians and around 60% of general practitioners knew the existence of the specialty and the importance of the primary dentition and examining them . The results correlate with similar studies showing more number of pediatricians than general practitioners, knowing about the specialty and importance of the primary dentition . Studies on oral examination by family physicians were not available but an american study showed that only 54% of pediatricians examined the oral cavity of more than half of the 0 - 3 year olds . Certain cranio - facial abnormalities can be diagnosed soon after birth, like pierre robin syndrome with the characteristics of glossoptosis and cleft lip and/or palate, lesions such as bohn's nodules and esptein's pearls being embryonic remnants and neonatal teeth that can make nursing difficult along with a risk of aspiration making examination of the oral cavity soon after birth a necessity . Evaluation of the knowledge of the specialty of pediatric dentistry, importance of the primary dentition and referral attitudes among pediatricians and family physicians it was noted that around 60% of the pediatricians and around 90% of the general practitioners felt that the parents will pose a barrier in the referral and importance of primary dentition . Studies on the relationship between parental attitude and its influence on the referral of patients were not available but age of the patient as an influencing factor for referral by pediatricians showed that practice that had infants or toddlers in the practice had lesser referral percentage . The knowledge and understanding of the health care providers [table 2] showed that nearly 70% of the pediatricians and only 26% of the family physicians could relate the importance of breast feeding and oral health . During breast feeding the infants oral muscles are exercised strenuously in suckling, an important influence on the thrust and growth of the mandible, in addition to imparting maternal immunological components . Appropriate, health - promoting practices, such as breastfeeding, should be encouraged by healthcare professionals . A study on children participating in the promotion of breastfeeding intervention trial, a cluster - randomized trial of a breast - feeding promotion intervention based on the who / unicef baby - friendly hospital initiative showed no evidence of beneficial or harmful effects of breast feeding at an early school age . What the pedodontist needs to emphasize is the oral hygiene, duration of breast feeding in association with a pediatrician and preventive methods to decrease the possibility of dental caries . Evaluation of knowledge of preventive practices and pediatric dentist's role in early intervention and special health care needs among pediatricians and family physicians there was decrease in the percentage for both groups with respect to the knowledge of the age of first dental visit, preventive care and knowledge of early childhood caries (ecc) with less than 50% of family physicians being aware of the same . Similar indian studies in showed that more than 90% of the pediatricians having knowledge of the same but information from family physicians were not recorded . Analysis of indian studies on the amount of oral health education received in the form of continuing medical education programs were not available yet most respondents received 2 h or less of preventive dental education during medical and specialty training . Pediatricians were better informed than family physicians in the areas of general dental knowledge and prevention counseling related to oral health . This could be the reason why this study revealed a low percentage of family physicians having knowledge of ecc . The site of implementation of oral health education seems to be an important factor where the most effective site was that of the physician's office itself . Children with ecc are three times more likely to develop dental caries in the permanent dentition . A study in united states has suggested the one year dental visit should be performed for all children in the lower socioeconomic status and can be elective for the middle and higher socioeconomic group, however when the risk factors are high the 1 year dental visit can be implemented . Pediatric dentistry as a specialty that provided early solutions to predictable orthodontic problems and its early role in cleft lip and palate as recognized by 50 - 60% of the pediatricians and around 50% of the family physicians . There are specific needs with regards to the child's oral development that can be identified in different stages of development . The dentist forms an important role in addressing the parent's concern of the aesthetics, early management involving preparing for surgery, management of pain, sepsis and when necessary dental extraction . The knowledge of effect of systemic disease on oral health had a higher response by around 53% of pediatricians and only 30% of the family physicians . Oral changes such as involvement of the temporomandibular joint and asymmetry of the mandible are seen in patients diagnosed with juvenile idiopathic diabetes and type i diabetes mellitus in children showed delayed dental development after the age of 10 with increase in periodontal disease and higher incidence of caries in those with poor metabolic control . Defects if the tooth structure such as enamel hypoplasia is seen in patients with renal disease and tetracycline used to treat infection cause intrinsic staining in hemodialysis patient . Extrinsic staining can be prevented by using medication in the form of capsules rather than syrups . Studies on oral health status of children admitted in a pediatric intensive care unit showed an significant increase in plaque accumulation and gingival inflammation between admissions citing the necessity for a better treatment regimen . Poor oral health on the other hand as seen in patients with chronic kidney disease may contribute to protein energy wasting, inflammation, infections and atherosclerotic complications, justifying a necessity for increased dental care . Pediatric dentists as trained professionals in handling handicapped children were acknowledged only by 47% of the pediatricians and only 29% of the family physicians . The importance of early referral to a pediatric dentist is necessary as the specialty involves patient control, caries preventive and management under general anesthesia, special gingival care involving children, especially under anti - convulsion therapy, dental trauma protection and treatment were the approach is quiet different than that of normal children as the patient cooperation does not allow fixed replacement and finally early intervention orthodontics such as a well timed extraction program to achieve a fairly acceptable occlusion . Only 27% of pediatricians and 17% of family physicians acknowledged the importance of the cariogenicity of syrups prescribed in dental practice . It has been noted that though antibiotics can have an effect on bacteria, their role is minor in caries prevention when compared to fluoride . A brazilian study revealed that around 56% of the medicated syrups had a high sugar concentration, with ph values below the critical value and high acidity values, all of them increase the cariogenic and erosive potential . The third part of the questionnaire [table 3] dealt with the knowledge level of the practitioners that only 27% of pediatricians and only 10% of general practitioners had received training in oral health and more than 90% of the practitioners were willing to undergo training in oral health . The most common barrier to participation in oral health - related activities in their practices was lack of training, which was cited by 41% . Less than 25% of pediatricians had received oral health education in medical school, residency, or continuing education . Studies have shown than the rates of preventive oral care in medical offices were not influenced by the cme courses . Increased practice volume in pediatric offices showed a greater rate of preventive dental services offered when compared to family physicians showing a lesser volume of pediatric patients . There seem to be certain barriers in adopting dental services, such as integrating the preventive dental services into the practice routines in the form of insufficient time to plan for change or provide services, practice procedures, and practice turmoil, which could be easily overcome by changes in practice timing along with training . Resistance by staff and colleagues was another obstacle and the incentive system seems to come over this . Applying fluoride varnish was the third obstacle as the physician does not have a working position as in a dental chair . This can be overcome by knee to knee position and provision of training materials and in - office training with a child patient for demonstration is suggested . Evaluation of the training received by the physicians and willingness to receive training among pediatricians and family physicians since the day of hippocrates, father of medicine, the physician and surgeon have made remarkable progress but there seems to be some missing in the relationship in the form of oral care, which should have been augmented by the dentist . The dental surgeon has undergone basic medical training including a thorough knowledge of head and neck anatomy; similarly the medical fraternity can be trained in the implementation of oral health . With the results obtained the following can be suggested . Collaboration between pediatric dentists and physicians for implementation of oral health education programs.promote oral examination of neonates by a pedodontist soon after birth.promotion of healthy referral practice maintaining ethics, even though the literature review shows a favor towards monetary benefits.enhancing out - patient and in - patient based continuous review of long term admissions and implementation of preventive procedures.promote the need for increased specialty dental care for special children.advise pharmaceutical companies to promote and convert to syrups with sugar substitutes.counseling sessions for to be parents . Promotion of healthy referral practice maintaining ethics, even though the literature review shows a favor towards monetary benefits . Enhancing out - patient and in - patient based continuous review of long term admissions and implementation of preventive procedures . Promote the need for increased specialty dental care for special children . The need for an increased awareness of oral hygiene habits and insufficient awareness among the physicians apparently warrants the common effort of both dentists and physicians for a better outcome.
Vascular endothelial growth factor (vegf) is currently considered the most important mediator of angiogenesis in physiological and pathological processes, including various eye diseases such as exudative age - related macular degeneration (amd). Ranibizumab (lucentis, novartis, basel, switzerland) is a monoclonal anti - vegf antibody fragment, designed and registered for intravitreal injections in treatment of neovascular age - related macular degeneration (amd). It acts selectively in locations where choroidal neovascularisation (cnv) forms, blocking and inactivating all known biologically active vegf isoforms . At the same time it tightens and stabilizes vessels . Because of these effects, retraction of subretinal fluid and intraretinal edema, which are signs of the degeneration process, the aim of this paper is evaluation of functional and anatomical results of intravitreal ranibizumab injections and of exudative amd treatment course in our 12-month observational study . After approval by the bioethics commission, 25 patients from the retinal clinic at the department of ophthalmology, military medical institute in warsaw were included in the study . Qualification criteria were as follows: (1) age of over 50 years; (2) active subfoveal dominantly classic neovascular amd, confirmed on fluorescein angiography (fa) (heidelberg engineering hra 2) and optical coherence tomography (slo oct oti), not treated previously; (3) overall lesion size not exceeding 12 optic disc diameters; and (4) baseline va of 2276 letters (early treatment of diabetic retinopathy study patients with the following conditions and past treatments were not qualified for ranibizumab treatment: (1) permanent structural foveal damage (subretinal scar or geographic atrophy); (2) macular hemorrhage exceeding 50% of lesion size; (3) retinal detachment; (4) vitreoretinal or filtration surgery, or transplantation of the cornea; (5) peripheral retinal photocoagulations within the last month; (6) macular photocoagulations; (7) cataract surgery within the last 2 months; (8) unstabilized glaucoma; (9) active infection of the eyeball or its protective apparatus; (10) past or active uveitis; and (11) significant degeneration of peripheral retina . The most important general exclusion criteria were cerebral stroke or myocardial infarction within the last 6 months, severe unstabilized hypertension, and unstable coronary heart disease . The mean age of patients was 73.238.55 years (range: 5888 years of age); 14 women and 11 men were treated . Treatment was performed according to the following schedule: each patient received 3 intravitreal injections of 0.5 mg ranibizumab at monthly intervals (saturation phase); further treatment was based on activity of the exudative degeneration process . After the end of the saturation phase, each month the patient s va was tested with the etdrs chart and oct was performed . The following re - injection criteria were employed: (1) loss of 5 or more etdrs letters compared to best result during the initial phase of treatment; (2) persistence or appearance of fluid under the retina or pigment epithelium, or intraretinal edema, on oct; (3) increase in central retinal thickness (crt) of at least 100 m compared to lowest crt during the saturation phase; (4) new macular hemorrhage; and (5) new cnv focus on fa . Each ranibizumab injection was performed in an operation theatre with aseptic protocol maintained during preparation of instruments, procedure field, anesthesia, the drug and its administration . Before and after the injection the patients were monitored for topical and general adverse events, and mean number of ranibizumab injections was counted . Mean va (etdrs letters, decimal scale, logmar) and mean crt were evaluated and compared at baseline, after the 1, 2 and 3 injection, and at 12 months . Existence of correlations between changes in va and retinal thickness in succeeding periods was tested . Anova testing was used for statistical analysis of changes in va and crt . Variation analysis with p<0.05 showed that mean va (etdrs letters, decimal scale, logmar) was significantly different at successive intervals . Mean best - corrected visual acuity (bcva) at baseline was 46.2816.38 etdrs letters (0.230.15, 0.730.27 logmar). After the first injection a significant improvement to 55.7216.24 letters (0.320.21, 0.580.29 logmar) was observed, and after the third injection the best va of 58.8013.79 letters (0.340.21, 0.540.27 logmar) was observed . At 12 months mean va was 56.8812.52 letters (0.310.19, 0.580.26 logmar) (figure 1). A deterioration of up to 5 etdrs letters was seen, and 32% achieved a significant improvement of 15 letters or more . Variation analysis at p<0.05 showed that mean crt differed at successive periods (figure 2). After the first ranibizumab injection it decreased significantly to 221.9660.85 m, after the third injection it was 200.8047.63 m, and after 12 months it was 213.1644.37 m . Frequencies of eyes with crt> 200 m were: baseline 100%, 1 month 48%, 2 months 40%, 3 months 40%, and 12 months 56% . Mean correlations between baseline average crt and baseline average va measured in etdrs letters (p=0.017) and in logmar scale (p=0.033) (figures 3,4) and between average crt after the third injection and average va in logmar scale after the third injection (p=0.047) (figure 5) were noted . The average number of injections in 12 months was 6 (48 injections in each patient). One of the most important clinical observations on ranibizumab, which should be referred to when discussing study results, is the anchor trial, in which patients with dominantly classic amd were qualified . The control group consisted of patients treated with photodynamic therapy (pdt) at 3-month intervals . In 90% of eyes, monthly intravitreal injections led to stabilization of vision at 24-month follow - up, while in the pdt group the result was 66% . In 41% of those patients improvement of 3 lines or more was observed, in 12% the improvement was 30 letters or more, and in the pdt group vision improved in 6% of patients . Mean va improvement in this group at 24 months was 11.2 letters . In the marina trial, which included patients with minimally classic or occult baseline va and lesion size were not shown to have a statistical influence on final functional results . There was no difference in mortality rate between the lucentis group and the control group . Ranibizumab therapy is indicated in all cases of exudative amd with involvement of the fovea, including perifoveal, provided the degeneration is active . In our observation presented here, only patients with classic form cnv were qualified, because this form of neovascular amd is uncommon, and in most studies patients with classic subretinal membranes constitute less than 50% of the study group . Baseline visual acuity is not a factor that should limit qualification for treatment in cases without irreversible damage of foveal function . Clinical trials have shown that even in cases of very low baseline va below 20/200, improvement of about 8 letters can be expected . Patients with active exudative sub- or perifoveal amd and good baseline va above 20/40 should be qualified for ranibizumab treatment due to a high potential for maintaining normal everyday functioning including reading and driving [710]. Both patients with poor bcva (22 etdrs letters) and with good va (76 letters) were qualified for our study . According to the marina and anchor studies, duration of degeneration is not decisive for treatment effects . In our study we additionally isolated a subgroup of patients with baseline va 2224 etdrs letters (4 cases). In this subgroup a significant mean improvement of 18.75 letters at 12 months was observed . Treatment of neovascular amd with ranibizumab usually begins with a saturation phase that consists of 3 monthly injections of 0.5 mg; our study proceeded according to this schedule . In this phase retraction of subretinal fluid, retinal edema and detachment of pigment epithelium correlates with va improvement . Fluorescein angiography is performed in individual cases of visual deterioration or new ophthalmoscopic lesions in the macula . Best effects have been achieved in the marina and anchor trials with monthly injections of ranibizumab; however, modified treatment schedules with fewer injections are being sought because they are cheaper and safer for the patient . In the pier study, after the saturation phase patients received quarterly injections of lucentis during an observation period of 24 months . The mean final va was 2.2 letters worse than baseline, while in the untreated group va deteriorated by 21.4 letters . These results formed the basis for the idea that the scheme of monthly injections can be modified with benefit for the patient . In the pronto trial, after the saturation phase, further injections were administered according to strict monitoring of topical state and changes in va, crt, oct and ophthalmoscopic macula image, and in some cases fa . Results of the pronto study (mean improvement in bcva 9.2 letters, 35% patients gained> 15 letters, mean 5.6 injections), comparable with marina or anchor, have made this kind of approach, which assumes stabilization and improvement of visual function with reduced number of injections, more and more common in retinological practice . In our study a mean of 6 injections, between 48 in each patient, were performed during a 12-month period and allowed an improvement 10.6 letters (32% of patients had improvement of 15 letters or more) and stabilization of va and crt parameters . In the pronto study the mean crt was 394 m at baseline and 216 m at 12 months . In our study, mean crt declined from 351.1274.15 m to 213.1644.37 m at 12 months, thus the results are comparable, but in the pronto study only 17.5% (7 eyes) had predominantly classic lesions . The sustain study also utilized the scheme of 3 saturation injections and further injections according to activity of the disease . In the sailor cohort 1 study, after the saturation phase, patients were monitored quarterly and reinjected if vision deteriorated by at least 5 letters compared to best result or if crt increased by at least 100 m compared to the smallest value achieved . After the saturation phase, significant improvement was observed, but final va deteriorated by 2.3 letters compared to baseline . These results prove that quarterly visits and reinjections are not sufficient for stabilization and maintenance of functional improvement . In relation to our observations, a clinical study by kumar et al reported the mean number of intravitreal ranibizumab treatment 5.62.3 in 81 patients with neovascular amd with similar treatment protocol to the pronto study . Frequency of predominantly classic lesions in this study was only 22%; 17.1% of all patients gained 15 letters or more (in our study 32% of patients achieved a significant va improvement). Mean bcva change at 12 months was + 3.711.1 letters . The subgroup analysis by kumar et al . Showed a mean change in va at 12 months of + 6.9 letters for classic no occult cnv, + 5.6 letters for predominantly classic with no occult lesions (in our study it was + 10.6 letters). Best results were noted in the rap subgroup, with mean va improvement of 8.9 letters . On our good results influenced a significant va improvement in the subgroup of patients with poor baseline va 2224 letters (16% of the group, at 12 months + 18.75 letters) and systematic control visits every month during 1-year observation which allowed us to notice each sign of the neovascular activity process . Bandukwala et al reported the effectiveness of intravitreal ranibizumab in a 94-patient group with neovascular amd . The mean number of injections was 5.1, with a mean of 9.4 visits in the 12-month period . There was a gain of 2.88 letters in all eyes, and a loss of 2.5 letters in the subgroup of patients who met the visual acuity inclusion criteria for the clinical trials . In this subgroup cohen et al confirmed this conclusion by results of their 1-year study in 124 eyes treated on a pro re nata basis after 1 or 3 initial intravitreal ranibizumab injections . The mean number of injections was 3.79 (range, 17), and the mean number of follow - up visits was 8.07 (range, 412) over a mean sd period of 526 weeks . Mean va standard deviation changed from 56.1514 to 56.8917 letters (va gain, + 0.7 letters). Cnv cases were of the classic type in 31 eyes (25%) and of the occult type in 93 eyes (75%). The results presented by cohen et al . Once again suggest that long - term regular follow - up is necessary for patients treated with ranibizumab to obtain and preserve significant visual gain, and not only to achieve visual stabilization . One of the aims of current clinical studies in patients with wet amd is to adapt the treatment to each individual to reduce the number of injections preformed . The results show great inter - patient variability in the number of injections needed, ranging from 1 to 23 over the course of 2 years [1921]. In a study by rothenbuehler et al . Initial treatment consisted of 1 ranibizumab injection; thereafter, all patients had follow - up examinations at monthly intervals as suggested by the marina and anchor trials . Retreatment was performed monthly if indicated based of cnv activity in oct, fa and ophthalmology examination with va evaluation . In spite of using only 1 initial dose of ranibizumab, but with systematic control visits each month, after 24 months 30% of 129 treated eyes gained 15 or more letters . Mean injection number per patient was 5.62.9 from baseline to month 12 and 4.33.8 from month 12 to month 24 . Arias et al reported a case series study of 90 eyes that were initially treated with 3 consecutive monthly intravitreal injections of ranibizumab, and thereafter follow - up visits were progressively spread out to a maximum of 8 weeks apart . Median va improved from 56 letters at baseline to 60 letters at 12 months, with significant reduction in foveal thickness . The mean number of injections was 4.4 and the number of visits was 8.0; 40% of patients received 3 injections and 60% received more than 3 injections . In this study no significant association was observed between va improvement and the number of injections (the same as in the pronto study). Like our study, arias at al confirmed that a flexible regimen with ranibizumab therapy is efficacious and safe in patients with neovascular amd, but reducing the burden of injections correlates here with reducing follow - up visits (fewer injections, control visits, and less effective in improving va than in our study). In a short 6-month study kloos et al reported no significant improvement in va for classic cnv (42/195 eyes) + 0.87 snellen chart lines in patients treated with repeated intravitreal injections of ranibizumab as needed . Better results were obtained in the occult or minimally classic lesions subgroup; 45% of eyes with predominantly classic cnv had received photodynamic therapies prior to the injections, and mr (szb reading chart) remained stable in 80% over 6 months . Rotsos et al described a retrospective analysis of the 50 patients with neovascular amd (classic form 10%) treated with ranibizumab . The mean number of injections was 4.7 per 12 month period, with 26% gaining 15 letters or more, and mean change in va was + 4.62.2 letters . It is difficult to compare those results with ours because of the small subgroup with classic cnv and heterogenous qualifications criterions (some eyes were treated previously). In our study 3d retinal topography full field 29.2 with high quality and high resolution frames we also performed radial scans and reviewed them for presence of subretinal fluid, intraretinal edema, fluid under the retinal pigment epithelium . We noted mean correlations between baseline average crt and baseline average va measured in etdrs letters (p=0.017) and in logmar scale (p=0.033) and between average crt after the third injection and average va in logmar scale after the third injection (p=0.047). Kumar et al did not observe statistically significant correlations between crt and va, finding only a slight increase in the number of patients with crt over 200 m at the 9-month visit corresponding to the drop in vision . In the pronto study, statistically significant correlations were found between the oct - crt measurements and va at months 2, 3 and 12 . Another strategy in the pronto study was to examine the association between oct changes at month 1 with va changes thereafter to determine if oct improvements could serve as a predictor of future va improvements . Statistically significant correlations were detected when the oct - crt measurements at month 1 were correlated with the va changes at month 2 . Fung et al described significant correlations between the decrease in crt at 1 month and subsequent improvement in va seen at months 2 and 3 . In addition, significant correlations were identified between the change in thickness at months 2 and 3 and va at months 2 and 3 . Did not observe significant correlation of morphological changes like subretinal fluid, intraretinal edema or sub - rpe fluid on the crosshair oct scans to the visual outcome at months 12 and 24 . After treatment with anti - vegf agents, the recurrence of subretinal fluid precedes the visual loss . The immediate effect of intravitreal ranibizumab on retinal anatomy, as seen by standard oct technology, was proven by fung et al ., who reported a resolution of sub- and intraretinal fluid . Described that ranibizumab rapidly diminishes the leakage activity of the neovascular net, and slower pace has a direct impact on the morphology of the rpe lesion as the subretinal lesion progressively flattens and the abnormal rpe area decreased in size during the initial treatment phase when visual function is positively affected in 80% of treated eyes . Visual recovery after resolution of macular fluid in wet amd likely depends on many variables, including chronicity of disease, viability of photoreceptors and the rpe, progression of the underlying dry abnormalities, and the presence of epiretinal membranes, rpe tears, and fibrosis . Used spectral domain - optical coherence tomography (sd - oct) to image cnv structure before and after anti - vegf treatment . Cnv (classic 16/78, occult 54/78, minimal classic 8/78) was imaged before and 4 weeks after anti - vegf upload in 3 intravitreal injections of ranibizumab; 59% of all post - treatment octs reveled complete dry retinal structures, 27% showed reduced edema, and 14% showed edema remaining unchanged . Macular thickness reduction was significantly enhanced, especially in cnv with classic components . In this study ranibizumab monotherapy was able to morphologically stop further cnv growth but in most patients did not lead to major regression of cnv, especially of its occult components . The issue of how to deal with the increasing number of intravitreal injections in everyday practice is very important . The main goal is to improve vision and maintain this improvement at a maximum over time, with a minimum of inconvenience for patients (mostly elderly) and the treating physician . At the moment, it seems the reduction of numbers of injections to 68 per year in the first year will maintain visual gain . Nevertheless, at the same time, monthly controls seem to be necessary to be able to react quickly to the smallest signs of deterioration this does not only mean deterioration in visual acuity, but also fluid increase in oct . It seems that if visual acuity is once lost, complete recovery is no longer possible . Treatment with intravitreal ranibizumab injections according to the scheme of saturation phase followed by re - treatment based on oct image and va, provides amd patients with a chance of stabilization and even improvement of topical state with a lower number of injections and preserved topical and general safety.
This study reports a rare patient of a rapid spontaneously resolving acute subdural hematoma . In addition, an analysis of potential clues for the phenomenon is presented with a review of the literature . A 1-year - and-2-month - old boy fell from a height of approximately 2 m. the patient was in a superficial coma with a glasgow coma scale of 8 when he was transferred to the authors hospital . Computed tomography revealed the presence of an acute subdural hematoma with a midline shift beyond 1 cm . Repeat imaging after 15 hours showed the evident resolution of the hematoma and midline reversion . Progressive magnetic resonance imaging demonstrated the complete resolution of the hematoma, without redistribution to a remote site . Even though this phenomenon has a low incidence, the probability of a rapid spontaneously resolving acute subdural hematoma should be considered when patients present with the following characteristics: children or elderly individuals suffering from mild to moderate head trauma; stable or rapidly recovered consciousness; and simple acute subdural hematoma with a moderate thickness and a particularly low - density band in computed tomography scans . A 1-year - and-2-month - old boy fell from a height of approximately 2 m and lost consciousness after he experienced head trauma . The patient was sent to a local hospital immediately . During the interval emergent computed tomography (ct), the patient vomited several times . 1a) revealed a right frontotemporal asdh that was approximately 9 mm in size with a subarachnoid hemorrhage in the longitudinal fissure and midline shift beyond 10 mm . He was in a superficial coma with the same gcs score that was documented in previous medical records . Both of the patient's pupils were equal in size 2 mm but exhibited a blunted response to light . 1b) showed a decrease in hematoma density and diffuse cerebral edema with obviously squeezed ventricles; the midline was still deviated compared with the first ct scan . Given his consciousness and imaging findings, intracranial pressure monitoring was preferred . Even though the laboratory features were not surgical contraindications, the boy's guardians refused the placement of an intracranial pressure probe or a craniotomy . General monitoring aimed to maintain sao2 at> 95%, mean systolic blood pressure at> 90 mm hg, temperature at <37c, and paco2 at 35 to 40 mm hg . Owing to the lack of an accurate intracranial pressure assessment, mannitol was administered empirically at 0.5 g / kg every 6 hours . The third ct scan (fig . 1c) revealed evident resolution of the hematoma and midline shift improvement, with only a subarachnoid hemorrhage present in the posterior longitudinal fissure 24 hours after the trauma . After he had received 2 days of conservative management, the boy regained consciousness . C) showed that the asdh was almost completely resolved on the fourth day after admission, without redistribution to the upper cervical spinal cord . (a) initial ct scan revealed right frontotemporal acute subdural hematoma (arrow to show), with sah in anterior longitudinal fissure and midline shift exceeding 10 mm . (b) a second ct scan showed the decrease of hematoma (arrow to show) with obvious diffusion cerebral edema . (c) a third ct scan was taken after 24 hours of the injury revealed an evident resolution of hematoma (arrow to show) and midline revert with only sah left in posterior longitudinal fissure . Ct, computed tomography; sah, subarachnoid hemorrhage . Magnetic resonance imaging on the fourth day of conservative management . Transverse (a), sagittal (b), and coronal (c) planes were performed which revealed acute subdural hematoma complete resolution without redistribution to upper cervical spinal cord . The following 2 possible explanations were proposed: the asdh was diluted and washed out by cerebrospinal fluid (csf) after the tearing of the arachnoid membrane and the hematoma was redistributed to the subdural space . Wong et al measured the hounsfield units of the subarachnoid space, which was close to normal, and confirmed the redistribution of a hematoma to the subdural space then to the subarachnoid space . The theory was based on the flow of liquefied blood, which was thought to be driven by the change in intraparenchymal pressure . In those patients without available definitive evidence of blood redistribution, csf flushing seems more likely . Based on table 1, rapid resolution occurred more frequently in children, less frequently in elders, and least often in middle - age individuals . In children and elders, asdh is often caused by mild or moderate injuries, such as falling or slipping . In elderly individuals, brain atrophy and a wide cavum of the subarachnoid space can cause an arachnoid membrane slit under external force . For middle - age individuals, well - developed brain tissue and narrow compensatory space results in an arachnoid membrane that is comparatively difficult to tear with a mild injury . Wen et al proposed that gcs was higher than 8 in patients who exhibited this phenomenon . However, in table 1, 7 of 18 patients exhibited a gcs score of less than 8, and the minimum was 5 . In the process of the clinical evaluation, the dynamic change in consciousness is a more heavily weighted factor to consider than the initial gcs score . According to a review of all patients (table 1), most of those patients regained consciousness quickly . Although some of those patients did not regain consciousness quickly, their conditions were stable . In our case, the patient's consciousness improvement was even slower than the time of resolution according to the ct scan . Thus, by comparing the initial gcs score after head trauma, we inferred that a rapid recovery or relative stabilization of consciousness is more persuasive in these patients . The evaluation of images from ct scans that were performed during different phases of our patient clearly demonstrated the rapid transition of hematoma density from a high density to a mixed density . Some authors agreed that the potential sign in early ct of a rapid resolution was the presence of a low - density band . In our patient's second ct scan (fig . 1b), it was observed that remarkable cerebral edema could squeeze, in some way, the diluted hematoma . Kuroiwa et al suggested that diffuse cerebral edema was necessary for the spontaneous resolution of a hematoma . We speculate that cerebral edema leads to resolution via both mechanisms, but it may not be the prerequisite for hematoma rapid resolution . Indeed, in most patients, the diffuse edema was not even observed (table 1). We propose that the slice or middle thickness hematoma (<1.5 cm) would be suitable in the consideration of rapid resolution . Lee et al reported a patient with a hematoma thickness that was larger than 2.5 cm, yet it finally converted to a chronic subdural hematoma . The presence of simple asdh without contusion appears to be associated with rapid resolution . We suspect that this effect is due to the mass effect of the contusion, which impeded the flow of csf in the limited space . This patient has been reported because of clear evidence of csf dilution and a flush effect that led to resolution of the hematoma, and the finding that a distinctly swollen brain probably accelerated the process . There is no need for the deliberate evaluation of the phenomenon as a coincidence during treatment . We suggest that asdh rapid resolution should be considered when patients present with the following characteristics: children or elders suffering from mild to moderate head trauma; stable or rapidly recovered consciousness; and simple asdh with moderate thickness and a particularly low - density band in ct scan images.
During october 2009october 2010, a total of 448 respiratory specimens were obtained from 448 patients (258 male patients and 190 female patients) with rtis and fevers in a virus surveillance system in osaka, japan (9). The mean sd age of the patients was 41.4 53.7 months (range <1404 months), and 351 (78.3%) were <5 years of age . Procedures for viral nucleic acid extraction and cdna synthesis have been reported (9). Pcr for detecting hrv and enterovirus was conducted by using evp4 and ol68 - 1 primers, which detected hrv and human enterovirus, respectively, in amplicons of 530 and 650 bp, respectively (7). Results showed 178 positive specimens (140 for hrv, 16 for human enterovirus, 7 for hrv and human enterovirus, and 15 for an unexpected amplicon of 600 bp). To identify the 600-bp amplicon, we sequenced viral protein 4 (vp4) and vp1 genes . Blast analysis (www.ncbi.nlm.nih.gov/) showed that these isolates had high identity with ev68 vp4 (98.5%99.5% with the pav25426868 strain [genbank accession no . Hm370293]) and vp1 (96.7%97.5% with the md021 strain [genbank accession no phylogenetic analysis using vp1 sequences (14 of 15 osaka strains were sequenced) demonstrated that osaka strains were clustered in 1 group and differed from previously reported strains (figure 2). Monthly distribution of enterovirus 68 (ev68) in osaka, japan, october 2009october 2010, bars indicate no . Phylogenetic tree of enterovirus 68 viral protein 1 gene sequences constructed by using a 927-nt sequence corresponding to nt sequence 23553281 in strain 3799, osaka, japan, october 2009october 2010 . Genbank accession numbers for strains used in this analysis were fermon, ay426531; 3799, ef107098; jpoc10200, ab601872; jpoc10290, ab601882; jpoc10373, ab601873; jpoc10378, ab601883; jpoc10396, ab601884; jpoc10402, ab601874; jpoc10404, ab601885; jpoc10412, ab601875; jpoc10441, ab601876; jpoc10445, ab601877; jpoc10471, ab601878; jpoc10573, ab601879; jpoc10616, ab601880; jpoc10618, ab601881; md021, ay426491; tx021, ay426495; mo00, ay426493; wi00, ay426494; mn98, ay426497; tx99, ay426498; md99, ay426499; tx03, ay426500; mn89, ay426489; ny93, ay426490; ev70, d00820; and ev94, dq916376 nucleotide and amino acid identities among 4 osaka ev68 (jpoc10 - 290, 378, 396, and 404 strains; nt 5017265 corresponding to the 3799 strain), fermon, and 3799 strains were determined . To determine sequences, we synthesized cdna by using specific primers and amplified 4 segments (nt 1601153, 5433391, 31324032, and 37477333 corresponding to the 3799 strain). The fermon and 3799 strains are the only ev68 strains for which complete genome sequences are available . The 3799 strain was isolated from a 6-year - old girl with pneumonia in 1998 (h. norder, pers . The sequenced region coded partial 5 untranslated regions (utrs) and all structural and nonstructural viral proteins (vp43d). Identities between strains were calculated by using bioedit version 7.09 (www.mbio.ncsu.edu/bioedit/bioedit.html) (table 2). * osaka strains, jpoc10 - 290, jpoc10 - 378, jpoc10 - 396, and jpoc10 - 404; vp, viral protein . Among osaka strains, nucleotide and amino acid sequences were highly conserved (nt identity 96.5%100% and aa identity 98.6%100%). In contrast, osaka strains had lower similarities with the fermon strain (nt identity 83.5%91.7% and aa identity 90.6%100%) than with the 3799 strain (nt identity 91.9%98.4% and aa identity 95.4%100%). When we compared individual viral proteins in osaka strains with those in the fermon strain, no gene except for vp4 showed> 90% nt sequence identity; gene 2b showed the lowest identity (83.5%85.8%). Regarding amino acids, <95% identity was observed in vp1, vp2, and vp3 in the fermon strain, and no genes with <95% aa identity were found in the 3799 strain in contrast with osaka strains . Moreover, no integration or deletion of nucleotides was observed in vp43d sequences among osaka, fermon, and 3799 strains . To clarify why ev68 osaka strain genomes were smaller than those of other enteroviruses and the ev68 fermon strain (7), we aligned the partial 5 utr sequences (nt 541820 corresponding to the fermon strain) of 4 osaka, fermon, and 3799 strains (figure a1). Results showed that the osaka and 3799 strains had deletions at nt 681704 and 717727 in contrast with the fermon strain . Moreover, a 1-nt deletion in osaka strains was identified at nt 641 in contrast with the fermon and 3799 strains . Because 14 patients with ev68 were detected during 20062009 (8), detection of 15 patients with ev68 during a 4-month period suggests an ev68 epidemic in the summer of 2010 in japan . Phylogenetic analysis with vp1 sequences showed that osaka strains differed genetically from previously reported strains . For precise analysis of osaka, fermon, and 3799 strains, results showed that osaka strains more closely resembled the 3799 strain than the fermon strain . Alignment of partial 5 utr sequences showed that osaka and 3799 strains had deletions in 2 regions in contrast with the fermon strain, and the amplicon was shorter than expected . The 5 utr of enterovirus contains an internal ribosome entry site (10) that is associated with translational efficiency and virulence of the enterovirus (11,12). Deleted regions of osaka strains appear to be in the flanking region between the internal ribosome entry site and an open reading frame (1). Detection of ev68 in numerous patients was reported in france during 2008 (4) and italy during 20082009 (13). Because this deletion was found in the 3799 strain in 1998, recent detection of ev68 in japan reported that serum samples from 281 pregnant women in finland in 1983, 1993, and 2002 had high titers of neutralizing antibody against ev68 (14). All ev68-positive patients in this study were <5 years of age and had lower respiratory tract inflammation . Seroepidemiologic studies in finland showed that most adults might have been previously infected with ev68 and therefore might have neutralizing antibodies (14). Increased detection of ev68, especially in infants and children this result indicates that ev68 can replicate in blood and may damage the central nervous system . Ev68 was detected in cerebrospinal fluid of a young adult patient with acute flaccid paralysis (5). Epidemiologic data for ev68 are lacking, and little information is available regarding virologic characteristics . If one considers results of phylogenetic analyses and nucleotide and amino acid identities, evolutionary changes might have occurred in ev68 . Our results show the potential role of ev68 infection in infants and children with rtis.
The term non - alcoholic fatty liver disease (nafld) refers to the spectrum of diseases characterized by fatty infiltration of the liver ranging from steatosis, steatohepatitis, or cirrhosis . Hepatic steatosis with or without hepatitis, in the absence of alcohol use, was first described by ludwig et al . And is referred to as non - alcoholic steatosis or non - alcoholic steatohepatitis (nash). Nafld is a common disease with an estimated prevalence in unselected population of developed nations around 2030% . The rapid rise in the incidence of the nafld might be explained by the recent epidemic of obesity and metabolic syndrome, which are manifested at hepatic level as nafld [35]. However, in 2 - 3% of patients, nafld can progress to nash that can eventually cause progressive fibrosis and lead to cirrhosis and related complications including hepatocellular carcinoma [3, 6, 7]. Once patients with simple steatosis develop nash, up to 50% of them could develop advanced fibrosis . A two - hit hypothesis was then proposed to explain the pathogenesis and progression of nafld, where the first hit causes accumulation of excess triglycerides in the liver leading to simple steatosis and the second hit causes the steatosis to progress to inflammation and fibrosis [9, 10]. The two - hit hypothesis was recently questioned as it was suggested that the hepatic accumulation of triglycerides in the liver might be instead protective towards further hepatic damage [11, 12]. Development of obesity or metabolic obesity, defined by isolated increase in visceral fat in people who are not obese, is often seen as the starting point for development of nafld, leading to the cascade of events ending in the formation of hepatic steatosis . Development of obesity or metabolic obesity is seen as the initial step in the development of metabolic syndrome and non - alcoholic fatty liver disease . Obesity is likely due to contributions from multiple factors including but not limited to impaired central appetite regulation, genetic predisposition, and contribution from dietary factors and lack of physical activity . Central nervous system plays a critical role in regulation of body weight via a negative feedback mechanism . Increase in body fat stores alert the appetite center in hypothalamus leading to appetite control and adipose tissue homeostasis . Insulin and leptin are considered as prime mediators of this mechanism . In overweight individuals, the amount of leptin in circulation is high but they develop resistance to leptin and so their appetite is not well controlled, leading to failure of the negative feedback mechanism [16, 17]. The molecular mechanisms leading to leptin resistance and its role in the development of obesity are discussed in detail elsewhere . While increased caloric intake definitely has a critical role in the development of obesity, there has been considerable interest about various dietary components and their relative contribution to the development of obesity . Increased fructose consumption has been shown as a risk factor for development of nash and that increased fructose consumption correlates with the severity of fibrosis in patients with nafld [19, 20]. The explanation is that fructose consumption leads to obesity or metabolic syndrome and that nafld is the hepatic manifestation but it is interesting to note that increased fructose consumption is an independent risk factor for development of fatty liver irrespective of metabolic syndrome [21, 22]. Patients with nalfd are shown to have increased percentage of dietary fat content and also get much lower percentage of their calories from fruits [23, 24]; not only total fat content in the diet but also the composition of fat has seen considerable interest in recent times . Current literature supports the fact that diet of patients with nafld might be high in saturated fatty acids and n-6 polyunsaturated fatty acids and low in n-3 polyunsaturated fatty acids and monounsaturated fatty acids [2527]. Though much of the focus has been on diets with high percentage of fats, diet, rich in synthetic disaccharides gut microbiota and its interaction with the consumed nutrients have also been the focus of research and microbiota could have a possible role in obesity by their influence on amount of nutrients absorbed . Several mechanisms were proposed including altered gut permeability and digesting the ingested polysaccharides thereby increasing the amount of energy absorbed . While low physical activity might not directly contribute to nafld in otherwise healthy patients, increase in physical activity coupled with weight loss has been shown to improve liver profile in overweight patients with chronic liver disease . Two types of adipose tissue are recognized in humans: brown adipose tissue and white adipose tissue . Brown adipose tissue, mainly found in neonates, helps with heat production and has a protective effect against hypothermia . White adipose tissue, present in adults, consists of adipocytes, endothelial cells, fibroblasts, leukocytes, and bone marrow derived macrophages . Instead, new research is pointing towards adipose tissue having a more complex endocrine function mediated by the production of numerous proinflammatory cytokines called adipocytokines [31, 32]. It should also be noted that not all white adipose tissue might be the same; increasing volume of visceral adipose tissue and their production of pro - inflammatory cytokines seems to play an important role in development of insulin resistance compared to subcutaneous adipose tissue . Adipocytokines produced by the adipose tissue include adiponectin, leptin, resistin, visfatin, tumor necrosis factor- (tnf-), interleukin-6 (il-6), monocyte chemoattractant protein-1 (mcp-1; also known as ccl2 or cc - chemokine ligand 2), plasminogen activator inhibitor-1, angiotensinogen, retinol - binding protein-4, and serum amyloid a [3438]. Adipocytokines are not exclusively produced by adipocytes but some, like tnf-, are mainly produced by the macrophages in the adipose tissue . Mcp-1 produced by adipocytes is a major factor contributing to macrophage recruitment to the adipose tissue . Adipose tissue in obese individuals is associated with increased macrophage activity, which is responsible for almost all of the tnf- and major part of the il-6 expressed by the adipose tissue . Thus, obesity is state of chronic inflammation characterized by abnormal cytokine (adipocytokines) production and activation of pro - inflammatory signaling pathways . The following sequence of events has been proposed: development of obesity leads to increased volume of adipose tissue, followed by increased production of mcp-1 by the adipocytes, which attracts more macrophages to the adipose tissue itself . Once the macrophages in the adipose tissue are activated, a self - perpetuating inflammatory cascade is triggered by secretion of pro - inflammatory cytokines like tnf- and il-6 . As noted above, the distribution of fat is also important in the pathogenesis of metabolic syndrome and visceral adipose tissue is considered a better indicator of insulin resistance and cardio vascular disease . This could be due to either release of greater amounts of adipocytokines by visceral fat tissue compared to subcutaneous tissue in obese individuals or could be due to the fact that visceral fat has direct access to portal circulation and thereby having stronger impact on liver . Sodium salicylate an antiinflammatory medication has been used to decrease glycosuria associated with diabetes many years before the discovery of association between type-2 diabetes mellitus and increased inflammatory markers . Since then more studies have shown increased levels of inflammatory mediators like c - reactive protein, interleukin-6, and plasminogen activator inhibitor-1 in patients with type-2 diabetes [4548]. Obesity is a proinflammatory state with high levels of circulating pro - inflammatory cytokines and diabetes is also a state of chronic inflammation; how are these two conditions related? The answer to this question was provided by a study that has shown that tnf- can induce insulin resistance in obese rodents and also that neutralization of tnf- can decrease the insulin resistance with resulting increased peripheral uptake of glucose . Since then similar findings of elevated tnf- were also found in humans with increased insulin resistance and impaired glucose tolerance [5052]. High tnf- levels can induce insulin resistance in animal models through the activation of i - kappa - b - kinase- (ikk)/nuclear - factor - kappa - b (nf-b) and jun n - terminal kinase (jnk) pathways . Jnk can cause insulin resistance through the phosphorylation of serine residues in insulin receptor substrate-1 (irs-1) [54, 55]. Ikk activation leads to activation of nf-b via transcription and sub - sequent increased expression of markers and mediators of inflammation causing insulin resistance . Increasing obesity will lead to increased production of adipocytokines like tnf-, il-6 that lead to perpetuating cycle of jnk, and nf-b activation leading to worsening insulin resistance . Detailed review of signaling pathways associated with insulin resistance due to inflammation was discussed elsewhere . The liver plays a key role in lipid metabolism; its role includes uptake and de novo synthesis of free fatty acids (ffas) followed by conversion of ffas into triglycerides by esterification . These triglycerides are then released into the circulation as very low - density lipoproteins (vldl) or stored as triglyceride vacuoles in hepatocytes . Ffas that are not esterified into triglycerides will be metabolized in the liver by -oxidation (figure 1). In nafld, there is disruption of this cascade of events since the amount of ffas delivered / synthesized in the liver exceeds its oxidative capacity . This leads to increased triglyceride synthesis and as the triglyceride synthesis continues to rise and exceed the amount that can be released as vldls, triglycerides accumulate in hepatocytes causing hepatic steatosis [58, 59]. This step of development of hepatic steatosis is considered as first hit in the pathogenesis of nafld [60, 61]. This raises the questions: what causes increased availability of ffas to liver, is it increased delivery or is it due to increased de novo synthesis of ffas in liver? What is the role of insulin resistance? Other than increased ffa availability, does disruption of other mechanisms like -oxidation or vldl synthesis contribute to hepatic lipid accumulation? As much as 59% of hepatic triglyceride content is derived from free fatty acids and only 26.1% of the hepatic triglyceride was due to de novo synthesis as shown in this study, where isotope tracers were used to track hepatic fat content . This increased delivery of ffas to liver is due to insulin resistance because insulin resistance increases the total serum ffas levels due to increased lipolysis in peripheral adipose [63, 64]. Increased expression of this pathway is seen in patients with insulin resistance and is implicated in pathogenesis of nafld . Defective oxidation of the ffas and dysfunctional vldl synthesis were also thought to be a key factor in pathogenesis of nafld . Though delivery of increased amounts of ffas beyond the capacity of liver metabolism seems to be the primary cause of hepatic fat accumulation, it should be noted that disruption of other pathways could have a role and more importantly that insulin resistance is implicated in most of these mechanisms [13, 68, 69]. As discussed earlier, increased visceral adipose tissue is a risk factor for development of metabolic syndrome and visceral adipose tissue insulin resistance in visceral fat leads to increased lipolysis and subsequent delivery of ffas to the liver increases in an exponential manner due to its direct drainage into portal circulation . Hepatic steatosis was considered as first hit in the pathogenesis of nafld but it later became clear that accumulation of triglycerides is actually protective and that free fatty acids are the toxic substances that lead to steato - hepatitis and fibrosis [71, 72]. Diacylglycerol acyltransferase 2 (dgat2) is an enzyme responsible for esterification of ffas into triglycerides; inhibition of triglyceride synthesis by genetically deleting this enzyme has reduced hepatic steatosis in mouse model but made fibrosis worse due to ffa toxicity . Interruption of triglyceride synthesis could be the initiating event for ffas - mediated lipotoxicity (cellular toxicity due to accumulated fat) in liver cells . As such, hepatic triglycerides are called the good fat and ffas are called the bad fat . Studies that looked at the composition of hepatic and circulating free fatty acids have revealed that patients with nafld have elevated levels of oleic acid (a monounsaturated fatty acid, mufa) and palmitic acid (a saturated fatty acid, sfa) [75, 76]. On the other hand polyunsaturated fatty acids are not shown to be toxic to hepatocytes and could be protective in patients with nafld [13, 77]. Further information on this topic was provided by experimental studies that looked at the role of stearoyl - coa desaturase-1 (scd1), the enzyme that converts sfa to mufa . Increased expression of scd1 leads to more mufa production, which was then incorporated into triglycerides and thus leading to well tolerated simple hepatic steatosis . But inhibition of scd1 leads to accumulation of sfa and subsequent development of hepatocytes apoptosis and steatohepatitis [74, 78]. So for disease progression in nafld, the type of ffa accumulated is as important or may be more important than the quantity of ffas accumulated in the hepatocytes . Apoptosis is a process of programmed cell death and is considered an important mechanism in the progression of nafld [8082]. Apoptosis is the key pathogenic mechanism noted in the biopsy specimens of the patients with nash and in the spectrum of nafld presence of apoptosis distinguishes patients with simple steatosis from patients with nash . The extent and severity of the apoptosis correlates with the degree of inflammation and fibrosis, so patients with higher apoptosis rates will have advanced stage fibrosis . Cytokeratin-18 fragments are markers for apoptotic hepatic cells and their circulating levels correlate with the severity of the fibrosis providing further evidence that apoptosis is an important feature of nash . Apoptosis mediated by ffas is called lipoapoptosis and the mediators of lipoapoptosis are further discussed here . Apoptotic pathways can be activated via extrinsic pathway mediated by receptors on cell surface or via intrinsic pathway mediated by intracellular organelles . Toll - like receptors (tlrs) are pattern recognition receptors that can identify pathogen - associated molecular patterns and in response, they activate the immune system via pro - inflammatory signaling pathways . Saturated fatty acids like palmitic acid can activate tlr4-mediated upregulation of nf-b with subsequent increased production of adipocytokines like tnf- and il-6 . Decreased expression of tlr4 in mutant mouse model is shown to be protective against development of nash . In an experimental dextran sulfate sodium (dss) colitis mouse model, mouse fed with high fat diet and dss had increased levels of bacterial lipopolysaccharides in portal circulation, increased expression of tlr4, and severe hepatic inflammation when compared to controls . Tlr4 might be the crucial link in the gut microbiota - liver axis related to progression of nash . Death receptors are cell surface receptors from the tumor necrosis factor family of receptors and play critical role in extrinsic apoptotic pathways . The death receptors and their ligands expressed in liver include fas, tumor necrosis factor receptor 1 (tnf - r1) and tnf - related apoptosis - inducing ligand receptor 1 and 2, trail - r1 and trail - r2, fas ligand (fasl), tnf-, and trail . In extrinsic pathway, death ligands activate their receptors forming adeath complex that in turn activates caspase-8 leading to apoptosis (caspases are death - inducing proteolytic enzymes). Reactive oxygen species (ros) are a group of free radicals derived from molecular oxygen, and oxidative stress refers to the cellular damage done by these free radicals . Ros are formed via oxidative reactions in intracellular organelles and mitochondria are a principal source of ros, but in a normal healthy cell, the levels of ros are very low due to various anti - oxidant defense mechanisms [94, 95]. In normal healthy subjects, mitochondrial -oxidation is the preferential way to dispose of the ffas by liver . But in nafld, there is an excess of ffas, and increased -oxidation by mitochondria leads to increased delivery of electrons to the electron transport chain causing overreduction of electron transport chain and formation of ros . Mitochondrial dna is vulnerable to damage by ros; increased generation of ros leads to damage of mitochondrial dna leading to mitochondrial dysfunction, which further potentiates ros formation . Intracellular stress caused by accumulation of ros leads to mitochondrial dysfunction resulting in release of proapoptotic proteins like cytochrome c into the cytosol . Cytochrome c then combines with apoptotic - protein activation factor-1 (apaf-1) and caspase 9 to form an activation complex called the apoptosome . Apoptosome activates the downstream caspases 3, 6, and 7 to complete the final steps of apoptosis . Mitochondrial dysfunction is considered the central pathophysiological process contributing to progression of nalfd to nash, and the quest to identify molecular mechanisms leads to the identification of lysosomal - mitochondrial axis in ffa - induced lipotoxicity and the potential role of lysosomal permeabilization in the progression of nash . In this study, liver cells were fed with high fat diet and observed in real time, lysosomal permeabilization and cathepsin b (a lysosomal protease) release in the cytoplasm occurred much earlier than mitochondrial dysfunction and cytochrome c release into the cytosol . Cathepsin b is also implicated in progression of liver fibrosis by its role in activation of hepatic stellate cells and aiding their differentiation into myofibroblasts . Endoplasmic reticulum (er) is an intracellular organelle with multiple important functions like protein synthesis, lipid synthesis, and so forth . When er is put under stress (er stress), it responds by a mechanism called unfolded protein response (upr). Upr is designed to protect er from the stress induced by various sources like viral infections, alcohol, or ffas . But when the duration of er stress is prolonged then upr might not be able to cope and leads to apoptosis [102, 103]. Further information about the role of er stress is addressed in this in vitro study where saturated fatty acid palmitic acid was able to induce er stress and lead to apoptosis of hepatic cells . Other mechanisms by which ffas can lead to apoptosis include mitochondrial dysfunction via c - jun n - terminal kinase (jnk) activation, pro - apoptotic protein bax - induced mitochondrial permeabilization, free cholesterol - mediated er stress, and ceramide - mediated apoptosis induced by death ligands like tnf / fas [74, 105] (figure 2). Insummary, impaired central appetite regulation, genetic predisposition, dietary caloric excess, and lack of physical activity contribute to development of obesity . Insulin resistance leads to increased lipolysis and exponentially high delivery of free fatty acids to liver . Accumulation of ffas leads to hepatic steatosis and ffa - mediated lipotoxicity that eventually progresses to fibrosis / cirrhosis (figure 3). In conclusion, nafld is increasing in prevalence and could become the most common cause of chronic liver disease in the near future in the western world . It is very important to understand the complex molecular mechanisms and the mediator involved to develop new therapeutic targets for this disease.
Retinitis pigmentosa (rp) is a hereditary disorder, in which the progressive loss of rod and cone photoreceptor cells occurs . Patients with rp generally experience loss of night vision (nyctalopia) in adolescence, side vision in young adulthood, and central vision (tunnel vision) in later life (14). About 14% of rp may accompany usher syndrome, which consists of hearing loss as well (1, 5). Retinitis pigmentosa is one of the most common causes of visual impairment in all the age groups with the prevalence of between 1/3000 and 1/5000 worldwide (3, 610). Retinitis pigmentosais a common and significant finding in iranian population as well . In studies done in cities of shiraz and shahroud, rp was found to be the 1 and the 3 most common cause of visual impairment, respectively (11, 12). Retinitis pigmentosa may be inherited as dominant autosomal, recessive autosomal, or x - related traits . However, it may sometimes be seen sporadically and without the family history (13, 14). Consanguineous marriage, especially first cousins marriage, has been reported to be the most common etiology of rp in iran (12). Patients with rp may develop a kind of a psychological disorder in a period of their life . Ignoring their mental disorder will deteriorate the rp course and finally result in their rejection by the society . Therefore, attempt to prevent, diagnose, and treat the psychological problems are necessary to improve the overall quality of life in patients with rp . To the best of our knowledge, this is the first study on assessing the common psychological disorders in patients with rp in iran . This descriptive, cross - sectional study was carried out between january 2009 and january 2010 on patients with rp . All the 939 patients who were members of iran rp center in tehran were recruited to the study through census sampling method . This study was approved by the ethics committee of nursing and midwifery school at islamic azad university, tehran medical branch as well as iran rp center . Then, the study goals were explained to the patients and a written informed consent was taken from each participant . Being older than 18 years, residence of tehran, member of iran rp center, and suffering from rp for at least one year were considered as inclusion criteria . Those with mental retardness or with a history of a diagnosed mental disorder prior to rp were excluded from the study . The necessary data were collected using questionnaires composed of two parts: the first part was about socio - demographic characteristics, including age, gender, marital status, education, occupation, income, type and location of accommodation, the family relationship between parents, the onset of rp, the history of rp amongst first - degree relatives, and cigarette, drugs, or alcohol consumption (14 questions). The second part composed of questions assessing the mental health and screening personality and psychosocial disorders, which were designed based on the modified minnesota multiphasic personality inventory-2 (mmpi-2) (71 questions). The mmpi-2 is an inventory assessing mental health in psychiatric and medical settings in adults above the age 18, which has been standardized for iranian population by mootabi and shahrami, and the validity and reliability of the questionnaire were confirmed (15). To examine the scientific reliability of the instrument, the cronbach s alpha index was applied data were analyzed using spss software (the statistical package for the social sciences, version 16.0, spss inc ., chicago, illinois, usa) through chi - square test to assess the relationship between socio - demographic characteristics and each mental disorder . The socio - demographic characteristics of the patients with rp are demonstrated in table 1 . As it shows, the majority of the participants were men (55.4%) in the age range of 26 to 35 years, and single (48.7%). Furthermore, most of the patients experience rp from their childhood (40.9%) and had the parental family relationship (60.7%). Socio - demographic characteristics of patients with retinitis pigmentosa based on mmpi questionnaire, patients with rp suffered from eight mental disorders with the following prevalence: obsessive compulsive disorder (39.3%), schizophrenia (38.1%), antisocial personality (37.6%), paranoia (36.7%), hypochondrias (35.3%), depression (31.2%), hysteria (26.9%), and hypomania (23.7%). The frequency of mental disorders in patients with retinitis pigmentosa the cronbach s alpha value was 0.8, which is satisfactory, and the power of study was 80% (if = 20%, 1- = 80%). Statistical analysis showed no significant relationship between obsessive compulsive disorder, paranoia, depression, and hysteria and background characteristics (p>0.05). A significant association was found between schizophrenia and onset of rp (p = 0.047). Furthermore, a significant association was seen between hypochondrias and educational level (p = 0.026) as well as income (p = 0.037), and smoking (p = 0.009). There was also a significant association between hypomania and marital status (p = 0.027). In our study, of 417 patients with rp, 231 (55.4%) were men, which is compatible with other studies . Because rp has x - related trait as well (13, 14), it is more prevalent in men while women are mostly carriers . Since the diagnostic value of mmpi-2 test decreases in subjects less than 18 years, we recruited patients above 18 years to our study . The most and the least of our patients have experienced rp from their childhood and infancy, respectively . This may be due to disability of infants to express the visual symptoms . Besides, since the specific amblyopic examination is done in childhood, the diagnosis of rp increases in that life period . Furthermore, because the parents are aware of the genetic transmission of rp to their children, they mostly take their child to an ophthalmologist for specific examinations, such as electroretinography . Of 417 patients in our study, this is in line with other studies, including those performed in iran (11, 12). Performed a study in shourideh educational center, which is the only educational center for blinds in shiraz, in the south of iran, and has students at different levels, from preschool to high school . Of 145 students, 65 (44.8%) had rp, which was the single most common cause of visual loss . Large proportion of these blind children (47.5%) had a positive history of consanguineous marriage in their parents (first cousins marriage) (12). Consanguineous marriage seemed to be the most probable cause of increased frequency of genetic etiologies of childhood blindness . Therefore, avoidance of consanguineous marriage may reduce the number of blindness caused by rp . In a study on 970 patients with rp, significant depression, anxiety, and phobia risk of depression in patients with rp was 25.1% more than that in the general population (17). Patients with rp that experienced depression in the same period had poorer visual function in comparison with those without depression (p<0.0005) (17). In 2009, rijavec and grubic reported a 28-year - old woman with usher syndrome (rp with hearing impairment), who developed psychological disorders, including eating disorder, psychosis, panic, anxiety, and obsessive compulsive disorder (18). In another study in 2012 on 26 children with usher syndrome, (23%) had a mental and behavioral disorder (19). John hopkins university followed up 50 patients with rp from 2007 to 2010 and reported that depression developed in patients with rp . The more depressed they were, the less visual function they had (17). In a study carried out on 144 patients with rp, relationship between depression and the vision - related quality of life was assessed . Therefore, it was suggested to diagnose and treat depression in order to enhance the overall quality of life in patients with rp (20). Depression was more frequent in patients with rp than those with glaucoma (p<0.0005) (21). Our results showed that depression was mostly reported in women with rp, especially those who were divorced and were in the age range of 46 to 55 years . Generally, depression is more prevalent in women . Besides, getting divorced and increasing age are themselves predisposing factors for depression as well (22). Yet therefore, patients should be addressed towards rehabilitating institutions that help them obtain new skills and return to their life . Furthermore, psychiatric counseling is required for patients with rp to accept and adjust to their condition . The findings showed that rp might lead to various mental disorders, especially obsessive compulsive disorder . Therefore, psychological support, which can be provided by either professionals or supportive patientsfamily, is often crucial in the course of rp . Further studies are required to evaluate the effect of orientation and training on the mental disorders in rp as well as comparing the psychological disorders in rp with other ophthalmological diseases . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) Have been completely observed by the authors.
Three hps cases were confirmed in misiones province, argentina, in the following patients: a 14-year - old boy from santa ana in november 2003, a 28-year - old man from leandro n. alem in december 2003, and a 12-year - old boy living in the dos arroyos locality of alem city . In january 2005, hps was confirmed in a 19-year - old girl living in pirapo, a rural area of itapua department, paraguay . Figure 1 shows the approximate geographic location of exposure sites for patients included in this study . Clinical manifestations in the 4 patients studied were similar to those reported for andv infections: fever, myalgia, headache, and vomiting, soon followed by pulmonary edema . Thrombocytopenia and hemoconcentration were reported, renal involvement was minimal, neither oliguria nor renal failure was observed in any case - patient, but all case - patients showed petechiae . The 4 hps case - patients had immunoglobulin m (igm) and igg antibodies to andv n recombinant protein by elisa (13) and all survived . These cases led us to investigate reservoirs for hantaviruses in misiones by using sherman live - capture traps (h.b . A total of 59 rodents were trapped at 2 study sites in misiones, where o. nigripes was the most frequently captured rodent (42 specimens), followed by akodon montensis (11 specimens). The rodent species was identified by morphologic features, in particular, qualitative external and cranial characteristics . Animals were tested for andv igg antibodies (table 1). The capture done in august 2005 in santa ana and leandro n. alem cities found 5 (11.9%) andv - positive o. nigripes rodents of 42 tested from this species . Misiones province, argentina, and eastern paraguay, where cases of hantavirus pulmonary syndrome have occurred and rodents were trapped for testing . * rt, reverse transcription . Before we discovered the case in itapua, we had conducted a serosurvey of rodents in 3 departments in eastern paraguay (table 1). Fifty - one rodent specimens were collected, of which 32 were sigmodontine rodents and 20 were akodon cursor . Only 1 a. cursor rodent obtained from the limoy biologic reserve, paraguay, was seropositive . Because of the diversity of akodontine rodents, precise diagnosis, based solely on morphometric characteristics, is not always possible . To confirm the morphologic identification of the a. cursor rodent, we compared the mitochondrial control region (fragment of 245 nt) with that of an a. montensis rodent used as a control . Mitochondrial dna sequencing of a. cursor from limoy found higher identity (94%) with an a. cursor from paraguay (af296264) than with a. montensis (90%). Positive voucher specimens were archived at the museo argentino de ciencias naturales and at the university of buenos aires . Pcr was performed on the 4 human blood samples and on the lung tissues of the 6 seropositive rodents . Initially, a substantial portion of the nucleoprotein n coding region of the s segment (nt 50954) and different fragments of the encoding region of the m segment: g1 glycoprotein (nt 41443), g1g2 glycoprotein (nt 1,7281,976), and g2 glycoprotein (nt 2,7152,941) were amplified and subsequently sequenced . Comparison of the viral 905-nt n fragment sequence from case - patient 1 showed the highest degree of identity, 90%, with lec (table 2). The strains from case - patients 2 and 3 showed little genetic variation between them and were 95% identical to arav from parana city, brazil (12), in the same fragment . Comparison of a g1g2 fragment available for juqv strain (nt 1,8671,976) with that from case - patient 2 showed a 93.6% identity . The g2 fragment from case - patient 3 was 95.6% identical to that of juqv . Thus, these results suggest that the strains from case - patients 2 and 3 are juqv, although they demonstrate that the strain called araucaria would also be juqv . * orf, open reading frame; lec, lechiguanas virus; orn, oran virus; bmj, bermejo virus; and, andes virus; ara, araraquara virus; cas, castelo dos sonhos virus; juq, juquitiba virus; ln, laguna negra virus; rime, rio mearim virus; anaj, anajatuba virus; riom, rio mamor virus; map, maporal virus; sn, sin nombre virus . Comparison done on 636 nt . Sequences from the 5 positive o. nigripes from misiones showed little variability between them and the n fragments were 98.3% identical to those in strains from case - patients 2 and 3 . The case - patient from itapua, paraguay, showed the greatest nucleotide identity (93.5%) with bmj lineage, in the n fragment; identity at the amino acid level was 99.3% . Another different strain was obtained from the a. cursor rodent captured in limoy reserve; this was the most distinct strain . The g1 fragment from this strain was compared with the closest related hantavirus and showed 67% identity with strains isolated from o. nigripes, case - patients 2 and 3, and lnv . All virus sequences from misiones and the itapua case form a monophyletic group together with andv lineages, nonpathogenic pergamino (prg) and maciel (figure 2), and casv and arav (done in a 643-nt parsimonious tree, data not shown). The other south american clade was formed with rio mearim virus and anajatuba virus from brazil, together with lnv, htn virus 007, and rio mamor virus from paraguay, peru, and bolivia, respectively, with a moderate support of 63% . Sequences from case - patients 2 and 3 from misiones grouped together with juqv (araucaria) to form a separate clade, since they were from the most divergent strains . The sequence from case - patient 1 grouped with lec lineage from central argentina . Different alignment parameters and phylogenetic methods produced the same results in trees with similar topology; however, bootstrap supports were moderate or low level for some lineages . The sequence from the a. cursor rodent was a quite genetically distinguishable virus lineage, separated and apart from choclo and maporal viruses . Phylogenetic relationships among the nucleotide sequences of the n protein of different hantaviruses from north america . A maximum parsimonious phylogenetic tree was generated on the basis of nucleotide sequence differences in the 904-nt region of the n gene open reading frame, which is available for south american strains by using phylip version 3.57c . Bootstrap values> 50%, obtained from 1,000 replicates of the analysis, are shown for the branch points . The following published s - segment sequences were included in the analysis (genbank accession no . ): hantaan (htn; u37768), seoul (seo; ab027522), prospect hill (ph; z49098), puumala (puu; x61035), black creek canal (bcc; l39949), bayou (bay; l36929), sin nombre (sn; l25784), new york (ny; u36801), el moro canyon (elmc; u11427), ro segundo (rios; u18100), cao delgadito (cdg; af000140), choclo (choclo; do285046), maporal (map; ay267347), htn-007 per (htn-007 per . Af133254), anajatuba (anaj; dq451829), rio mearim (rime; dq451828), ro mamor bolivia (riom; u52136), laguna negra (ln; af005727), araucaria (juq; ay740633), maciel (mac; af0482716), pergamino (prn; 482717), andes (and; af324902), oran (orn; af028024), hu39694 (hu39694; af482711), bermejo (bmj; af482713), lechiguanas (lec; af482714). Hps is an emerging disease in south america, and investigations strengthen the belief that the disease is underestimated . Despite being surrounded by hps - endemic countries, two pathogenic hantaviruses that cause hps have so far been proven to circulate in misiones: lec and juqv . We confirmed o. nigripes as the reservoir species associated with 2 juqv cases in misiones . In eastern paraguay, itapua department, bmj lineage produced hps . Lec was characterized originally from an o. flavescens mouse trapped in the rio de la plata river, an area where several hps cases had occurred (6)., no akodon - borne hantavirus has been reported to be associated with cases in south america . A. azarae is the most abundant sigmodontine species widely distributed in rural and peridomestic habits of central argentina . In buenos aires province, prg was characterized in a. azarae populations . Despite the absence of reported hps cases associated with this species in the studied area, we characterized a distinct akodon - borne hantavirus at the biologic reserve of paraguay, although we did not investigate whether this virus can produce illness . A prior study analyzed a collection of sigmodontine rodents from the major biomes of paraguay where 1 a. montensis and 2 o. nigripes were positive for viral rna (15). Precise identification of source populations in the reservoir and collection of quantitative data on their relative contribution to hantavirus transmission will be essential for disease control in the 3-country frontier.
The presence of butterfly rash is typically a sign of systemic lupus erythematosus (sle), but it can also signal a dermatologic disorder . Typically, butterfly rash appears in a malar distribution across the nose and cheeks . The case report presented here is of a 40 year old female with bright red discoloration of the skin involving the malar region bilaterally with the bridge of the nose and chin along with typical bright red colored swollen gingiva with shallow periodontal pockets and loss of attachment . Both the lesions exacerbated and relieved almost simultaneously which led us to correlate this dermatological condition to oral lesion . A 40 year old female reported to the department of periodontics, institute of dental sciences, bareilly, with the chief complaint of swelling and bleeding from gums associated with burning sensation since last two years and bright red discoloration of the skin involving the malar region bilaterally, bridge of nose and chin . Extraoral examination revealed malar rash involving the chin accompanied by yellowish white pustules scattered along the discoloration . The discoloration appeared first on cheeks, followed by nose and finally the chin as shown in figure 1, 2a, 2b, 2c, 2d . The discoloration was followed by acne, followed by pustules and finally the scar which healed by itself followed by its reappearance after 10 - 15 days . Bright red discoloration of the malar region and chin histopathologic picture showing inflammatory cells, blood vessels and connective tissue stroma intraoral examination revealed bright red discoloration of the gingiva involving the marginal, interdental and attached gingiva extending from #14 to 22 and #43 to 31 and mild generalized inflammatory enlargement was noticed [figure 2]. Detailed history from the patient revealed that the bleeding from gingiva was insidious, mild, intermittent, occurred only while brushing the teeth and aggravated on eating hard fruits . It appeared first in the interdental region and then progressed to involve the other parts of gingiva . Swelling increased in size gradually, decreased thereafter two months for about 15 days, then regains its original size . Radiographic findings [figure 3] showed features of chronic generalized periodontitis with horizontal pattern of bone loss and angular defect at some sites . Opg - features of chronic generalized periodontitis with horizontal pattern of bone loss and angular defect at some places laboratory investigations carried out included routine blood investigations, le cell phenomenon, ana studies (antinuclear antibody studies), anti ds dna (anti double stranded dna). Incisional biopsy of the specimen illustrated the presence of connective tissue stroma with eroded epithelium . As the intraosseous lesion was interfering with mastication and extraoral with aesthetics, the patient was subjected to phase - i therapy (oral hygiene instructions, scaling and root planning), antibiotic (doxycycline) was prescribed for five days followed by tapered dose for two days, mouthwash (listerine) for 1 month, topical corticosteroid (betamethasone) for extraoral application 3 times a day for 4 weeks . When the patient did not respond to the, systemic corticosteroids were administrated (tab wysolone 5 mg for 15 days). She was asked to wear the sunscreen with a sun protection factor (spf) of 15 or higher to protect her face from sun, avoid irritating the facial skin by rubbing or touching it too much, avoid the use of facial products that contain alcohol or other skin irritants and the use of non - comedogenic products was recommended . Post - treatment: disappearance of acne, pustule, reduction in erythema after one month: edema disappeared, burning sensation reduced, persistent erythema over #12, 11 butterfly rash is a red, flat facial rash involving the malar region bilaterally and the bridge of the nose . It is most suggestive of systemic lupus erythematosus, but it might be seen in variety of other dermatologic diseases such as erysipelas, rosacea, seborrheic dermatitis, sarcoidosis, polymorphous light reaction etc . Rash may appear as a prominent, non - scaling, intermittent erythema limited to lower half of the nose or including the chin, cheeks and central forehead . The malar rash often appears or gets worse after sun exposure (photosensitivity) or stress that causes an increase in the circulation to the skin . Sometimes the butterfly rash appears on other parts of the body as well, usually on the trunk, arms or legs . The reason the malar rash is shaped like a butterfly is because it follows the angle that the uv rays land on skin . It is caused by a malfunctioning immune system, which causes the body to attack healthy tissues in the skin . The present case is of a 40 year old female who presented with a butterfly shaped rash involving the chin and the forehead along with gingival manifestations . The intraoral clinical picture could be suggestive of an inflammatory lesion but the picture did not improve completely after phase - i therapy . On the basis of the clinical findings, the tentative diagnosis of lupus erythematosus was made, with the differential diagnosis of erysipelas, polymorphous light reaction, rosacea, seborrheic dermatitis, and sarcoidosis . In the present case there was no systemic involvement and special investigations like ana (anti - nuclear antibody) and anti double stranded dna were negative so sle was ruled out . There was no history of previous throat infection, fever and leukocyte count was within normal limits, thus no evidence of erysipelas . The extraoral appearance was neither patchy nor thick adherent crusts, so seborrheic dermatitis was also ruled out . The patient presented with persistent redness, presence of pustules, absence of black heads, burning and stinging sensation along with gingival enlargement and discoloration which signaled rosacea . Management of the dermatological disorder associated with oral involvement is often a complex undertaking and requires a joint expertise and communication of clinicians to provide the patient with an optimal treatment plan based on scientific rationale . All that glitters is not gold similarly all the butterfly rashes are not lupus erythematosus so a dentist should look into and treat accordingly.
Cancer cachexia is associated with increased mortality and morbidity in cancer patients . By international consensus, cancer cachexia is proposed to be a multifactorial syndrome defined by an ongoing loss of skeletal muscle mass with or without loss of fat mass that cannot be fully reversed by conventional nutritional support and leads to progressive functional impairment . A recent review reported elevated lipolysis to be the major reason for fat loss in cancer cachexia [4, 5] although the underlying mechanisms are undefined . As fat loss precedes muscle loss, associates with shorter survival [6, 7], and is variable with respect to timing and intensity in various cancer populations . Therefore, identification and validation of markers of fat loss are crucial not only for a better understanding of mechanisms, but also to identify fat losing cancer patients who will subsequently develop cachexia . Effective management of cancer cachexia is restricted to early identification of the syndrome; therefore, biomarkers are vital for development of appropriate therapeutic interventions to achieve better outcomes for individual cancer patients . Adipose tissue (at) is an active secretory organ, composed mainly of adipocytes and nonadipocyte cells such as inflammatory cells, immune cells, preadipocytes, and fibroblasts . Adipokines are proteins synthesized and secreted from adipocytes which act both locally and distally, contributing to whole body lipid metabolism [9, 10]. In pathophysiological conditions like cancer, macrophage infiltration into at increases [11, 12], leading to alterations in adipokine production affecting adipose tissue mass and function . Local adipokines produced by at, circulating cytokines, and lipid mobilizing factors are collectively involved in adipose atrophy in cancer cachexia [13, 14]. Considering adipose tissue as a metabolically active organ as well as the relationship between fat loss and shorter survival in cancer, early identification of fat losing patients may increase the opportunity for therapeutic management of cachexia . A biomarker is a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process or of a condition or disease . . Biomarkers indicate normal biologic processes, pathogenic processes, or pharmacological responses to a treatment . Biomarkers in the oncology setting, identified using high - throughput sequencing, gene expression arrays, and mass spectroscopy, are classified into prognostic, predictive, and pharmacodynamic categories [1820]. Prognostic biomarkers provide information about likely outcome of a disease, regardless of treatment, whilst predictive biomarkers assess the effect of a particular treatment . Ideal biomarkers are easily accessible, available, specific and sensitive, noninvasive, inexpensive, consistent, safe, and easy quantifiable in a biological fluid or clinical sample . Levels of the biomarker should not overlap between controls and patients while significantly relating to the outcome of interest using appropriate statistical analysis . While it seems important to identify a prognostic biomarker of cancer cachexia - associated fat loss, no ideal clinical biomarker has been defined yet, which demonstrates a need to identify and subsequently validate potential biomarkers in independent studies . Studies focusing on adipose tissue have identified leptin, free fatty acids (ffas), and glycerol in plasma as indicators of fat alterations in health and diseases . On the other hand, adipokines including inflammatory cytokines such as interleukin-6 (il-6) and tumour necrosis factor- (tnf-) as well as zinc-2-glycoprotein (zag) have also been associated with weight and fat loss in cancer . Therefore, circulating levels of these factors may represent new noninvasive prognostic biomarker of adipose atrophy and targets in the detection and management of fat loss in cancer . One of the major obstacles to identify reliable biomarkers of fat loss in cancer cachexia is variation between studies in how fat loss is assessed . Body mass index (bmi) is frequently used as a clinically accessible measure of human body composition . However, as bmi does not distinguish between fat and fat - free mass, its utility in the settings of fat loss in cancer cachexia is limited . Various methods including bioelectrical impedance analysis (bia), dual - energy x - ray absorptiometry (dexa), magnetic resonance imaging (mri), and computed tomography (ct) scan analysis have been applied to assess body composition in cancer population . Ct image analysis, as the gold standard for body composition assessment in cancer patients, has an ability to discriminate and precisely quantify different adipose tissue depots . Many patients have repeated scans over the cancer trajectory enabling assessments in the same individual over time . Application of body composition assessment in the cancer setting has focussed primarily on lean body mass . The studies that do exist reveal loss of adipose tissue as cancer progress [25, 26]. However, further studies are required to establish the timeline and pattern of fat mass alterations in different adipose tissue depots during cancer progression . Moreover, the majority of studies assessing fat mass focus on gastrointestinal cancer patients; there remains a gap in knowledge related to other malignant tumours . Finally, timing of ct scans differs between patients and scans may not be available over a specific time points demonstrating the need for other important prognostic biomarkers of fat loss . Overall, gaps remain related to the association between fat mass alterations assessed by ct scans and circulating markers of fat loss . This article reviews current knowledge around potential prognostic biomarkers of fat loss in cancer which may identify fat - losing cancer patients who would benefit from early therapeutic interventions to improve outcome of cancer patients . Possibilities and potential to apply these markers as prognostic biomarkers of fat loss will be discussed . Serum levels of cytokines associate with clinical features of cancer cachexia such as weight loss; however, no study has specifically assessed the association between serum cytokines and the extent of fat loss in cancer patients . Inflammatory cytokines, such as il-6 and tnf-, are produced by tumours and by nonfat cells residing in at in addition to adipocytes . Plasma levels of inflammatory cytokines are elevated in cachexia and are thought to promote adipose atrophy in animal and human models of cachexia . Pathways of adipose tissue metabolism evoked by il-6 and tnf- include inhibition of lipoprotein lipase mrna expression and activity which prevents fat cells from taking up fatty acids from lipoproteins [28, 29]. These cytokines stimulate hormone sensitive lipase (hsl) and adipose triglyceride lipase (atgl) activity [30, 31], leading to elevated lipolysis . Tnf- has been reported to prevent preadipocyte differentiation and inhibit expression of lipogenic transcription factors . Tumour presence has been associated with elevated serum il-6 and tnf- in mice bearing the lewis lung carcinoma or b16 melanoma cells compared to controls . In humans, data regarding the role of tnf- in cancer - associated wasting measuring tnf- in plasma is challenging due to short half - life and transient nature . Further, the sensitivity of assays used to measure plasma tnf- is variable, making comparisons between studies limited . On the other hand, tnf - r1 and tnf - r2 (soluble tnf- membrane receptors) have been applied as serum markers of tnf- activity due to their longer half - life and greater stability . A comprehensive review of clinical factors associated with cachexia showed little evidence for the association between serum tnf- and weight loss in cancer, while several studies report an association of plasma il-6 but not tnf- with cachexia - associated wasting rather than cancer per se . Serum il-6 levels were higher in fat losing gastrointestinal cachectic cancer patients compared to weight stable and noncancer controls . However, no changes in mrna expression or secretion of il-6 and tnf- from sat were observed . This finding was confirmed in another study showing that circulating il-6 levels were higher in weight losing non - small - cell lung carcinoma patients compared to weight stable cancer patients . Adipose atrophy has been associated with elevated il-6 signalling in a preclinical model of cancer cachexia . In patients with gastrointestinal cancer, plasma il-6 levels significantly correlated with the presence of tumour and increased with each progressive stage of cancer . Il-6 has been reported to be involved in early stages of cachexia [42, 43] and a study conducted in patients with mixed tumor types showed il-6 levels gradually increased during early stages of cachexia followed by rapid increase prior to death . In contrast, a study in 61 patients with advanced cancer showed no correlation between il-6, tnf-, and weight loss . Although circulating il-6 levels were higher in cachectic mice compared to controls, il-6 receptors deficient (il-6-r - ko) mice were partially protected so other cytokines may involve in cachexia - associated wasting . Moreover, a study published in 2012 reported that other cytokines, such as il-1 but not il-6, may be better indicator of cachexia features such as weight loss and body composition alterations . Collectively, evidence would suggest that inflammatory cytokines are involved in at depletion in cancer [13, 36, 42]; however, plasma concentrations may represent the presence of a tumour rather than cachexia - associated adipose atrophy per se . Future studies are required to assess changes in adipose tissue depots, both visceral adipose tissue (vat) and subcutaneous adipose tissue (sat) over the disease trajectory using validated body composition assessment tools and correlating those to changes in circulating cytokines . Given that there could be various sources of cytokines contributing to plasma levels, the transient nature of cytokines, as well as the cost associated with cytokine measures, the application of plasma measures of cytokines as biomarkers of adipose tissue atrophy in the clinical study is likely limited . Moreover, the ability of cytokines to evoke cancer cachexia depends on tumour type and the complex response within a network of mediators, rather than a single cytokine [47, 48]. Major gaps remain regarding the association between plasma cytokine levels and fat loss, clinical ranges of abnormal measures, and method sensitivity . Leptin regulates body weight by activating the anorexigenic neuropeptides and inhibiting the orexigenic neurons such neuropeptide y (npy) [50, 51]. Besides body weight and fat mass regulation normally, a lower plasma concentration of leptin is associated with higher npy secretion; however, npy pathways have been reported to be dysfunctional in anorectic tumour - bearing rats . Many factors influence leptin synthesis and secretion in adipocytes such as insulin, tnf-, glucocorticoids, reproductive hormones, and prostaglandins [54, 55]. In humans, a higher concentration of serum leptin in obese individuals is associated with increased fat mass and cell size . Serum leptin is considered to be an accurate, reliable, and highly correlated measure of total body fat . In healthy subjects, elderly adults, and obesity, plasma leptin levels have been shown to be a precise measure of adiposity . A relationship between low fat mass and low plasma leptin levels has also been reported in cancer patients [5967]. Advanced gastrointestinal and lung cancer patients experiencing cachexia - associated adipose atrophy exhibited hypoleptinemia [6769]. On the other hand, breast and gynaecological cancer patients exhibited elevated plasma leptin levels that related to the elevated levels of sex hormones and receptors, rather than cachexia per se . Circulating leptin concentrations have been used as an indicator of fat mass; however further studies are required to examine changes in leptin concentrations that occur throughout the disease trajectory and relative to body fat mass alterations . Longitudinal studies that employ a precise measure of body fat would enable determination of whether changes in plasma levels of leptin change proportional to fat mass alterations . An added level of complexity is that leptin is secreted by both vat and sat, with sat contributing the majority of leptin to plasma due to its larger contribution to overall body mass . Therefore, measures of changes in leptin concentrations over time do not currently represent the type of fat being lost or gained . Comparison between studies is limited by different assay sensitivities and how leptin values are reported as total, free, or bound leptin . Further, factors such as the type of cancer, bmi, and sex and age influence serum leptin concentration, as reported in adolescents, also need to be considered in study interpretation . Low leptin concentrations could be considered a result, not a cause of cachexia, which significantly relates to adipose atrophy and low fat mass in cachexia . Studies indicate elevated lipolysis to be the main cause of fat loss in cancer [4, 5, 7275]. During at lipolysis, ffas and glycerol molecules are produced by the action of lipolytic enzymes such as atgl and hsl, which hydrolyze stored triglyceride . Adipose atrophy has been associated with elevated activity of atgl and hsl in human and animal models of cancer [35, 40, 46]. Elevated lipolysis produces higher plasma glycerol in cachectic cancer patients compared to healthy subjects or weight - stable controls . Lipolytic activity was assessed in 13 cachectic and 14 weight - stable cancer patients by assessing circulating glycerol levels (mol / l / kg body fat) as an indicator of in vivo lipolysis . Cachexia was defined as> 5% weight loss over 3 months or> 10% within the previous 6 months . Body fat mass, assessed using bia, showed lower body fat (% and kg) in the cachectic group compared to weight stable patients . Elevated levels of plasma glycerol, ffas, and higher expression of genes involved in energy turnover pathways and oxidative phosphorylation revealed increased lipid mobilization from subcutaneous adipose tissue in the cachectic group . These results support those of agustsson et al . Who showed plasma glycerol and ffas to be higher in newly diagnosed gastrointestinal cancer patients with cachexia who had low body fat mass (kg), assessed using ct images, compared to the weight - stable group . Higher plasma glycerol and ffas in the cachectic group positively correlated with percent weight loss and negatively correlated with visceral adipose tissue area . Plasma glycerol values in cancer cachectic patients have been reported as mol / l or mol / l / kg body fat [4, 5, 72, 76]. Interestingly, studies focusing on lipolytic activity in cancer cachexia report a narrow range of plasma glycerol for cachectic patients between studies [4, 5, 72, 76], strengthening its use a potential biomarker . Plasma glycerol has been reported as 6.2 2.7, 6.9 1.3, 7.0 4.3, and 9.8 2 (mol / l / kg body fat) in cachectic patients compared to weight stable cancer patients reported at 3.1 0.7, 3.9 0.6, 3.4 1.6, and 3.3 0.3 (mol / l / kg body fat). Postabsorptive whole body lipolytic rate, assessed by glycerol infusion technique, revealed basal levels of plasma glycerol to be higher in a cancer group compared to controls . While lipolytic rates were similar, glycerol clearance rate varied between the two groups and contributed to higher glycerol levels . Although preillness weight loss ranged from 0 to 20% in cancer patients, the same results were obtained when data was corrected for body weight . Despite the use of plasma glycerol as an index of whole - body lipolysis, caution lipolysis results in the release of fatty acids and glycerol from adipose tissue, with glycerol being a better index of lipolysis as ffas liberated by lipolysis may be reesterified within adipose tissue . At has very low glycerol kinase activity, and glycerol released by lipolysis enters into the bloodstream . However, lipolytic activity is not specific to adipose tissue and occurs also from intermuscular triglyceride stores and plasma lipoproteins . Glycerol concentration may indicate that lipolysis occurs in sat as glycerol released from visceral adipose tissue lipolysis enters the liver via the portal vein . Therefore, plasma concentrations of glycerol reflect the balance between glycerol release by lipolysis (predominantly adipose tissue) and clearance of glycerol by liver and should be interpreted with caution . Zag is a protein discovered in human plasma that has been associated with presence of several types of carcinomas such as breast, prostate, and lung [8385]. Elevated serum zag, as a routine and reliable measurement, may apply to early diagnosis of cachectic cancer patients with adipose atrophy . Zag has been considered as an adipokine involved in lipid metabolism in adipose tissue [87, 88]. Both in vivo and in vitro studies have shown that increased zag expression in adipose tissue is associated with increased lipolysis and subsequent fat and weight loss [89, 90]. The exact mechanism by which zag participates in fat loss in cancer is not known . Zag may induce lipolysis through activation of -adrenoreceptors [89, 91] and elevated hsl activity [92, 93]. Although the mechanism behind zag regulation in at is still unknown, glucocorticoids have been suggested to stimulate zag expression in at . Increased plasma cortisol levels in cachectic tumor bearing mice and in cancer patients have been associated with higher at zag expression and elevated lipolysis . This implies that, in cachexia, glucocorticoids may induce lipolytic activity through an increase in zag expression [94, 96]. There is discrepancy in the association between circulating zag levels and weight or fat loss in various conditions . Data on serum zag levels in obesity are inconsistent, being reported as either increased or decreased which positively and negatively correlated, respectively, with bmi . Elevated serum zag levels have been observed in chronic heart failure and haemodialysis patients suggesting zag to be a marker of fat catabolism . In contrast, two studies in cancer patients [77, 92] demonstrated that plasma zag levels may not be a good biomarker of cachexia - associated features such as weight and fat loss . Twenty - five gi cancer patients underwent curative abdominal surgery and were categorized as cachectic or weight stable . Cachexia was defined as unintentional weight loss of more than 5% during the previous 6 months . Mrna and protein levels of zag in subcutaneous adipose tissue were higher in cachectic cancer patients compared to weight - stable cancer patients which significantly correlated with fasting serum glycerol levels and weight loss . In this study, however, there was no significant difference in circulating zag levels between cachectic and weight stable cancer patients . Production of zag by tumours and nonadipose tissue, such as the liver, may also affect zag plasma levels . This result is consistent with rydn et al . Who report that zag is a locally produced factor, promoting at lipolysis, but not secreted predominately to circulation . Therefore, circulating levels of zag are not likely to relate to fat loss in cancer cachectic patients but instead may mediate local lipid mobilising action in adipose tissue . Patients with advanced cancer frequently suffer weight and fat loss . Accelerated loss of adipose tissue is associated with shorter survival, reduced quality of life, and decreased muscle mass during cancer progression . Due to the role of adipose tissue in mediating human metabolism, identification of prognostic biomarkers of fat loss in cancer may help to identify fat losing cancer patients for early therapeutic interventions, improved survival, and prevention of muscle atrophy in cancer patients . No studies in cancer have identified a prognostic biomarker of fat mass alterations nor have the sensitivity, specificity, and reproducibility of potential indicators been assessed in the neoplastic state . Inconsistency in the literature may be due to varying sensitivity of assays used to measure plasma levels of mediators, heterogeneity of patient populations and treatment, and various body composition assessment methods . Inflammatory cytokines appear to be mediators of cachexia - associated features such as fat loss [13, 36, 42]; however, they do not fulfill several components of biomarker criteria . Relationship between circulating cytokines and degree of fat loss in cancer has not been assessed . Zag in plasma has been suggested to indicate the presence of some type of tumours, and in at, zag can act locally to modulate lipolysis . Literature regarding the potential of plasma zag to be a biomarker of fat loss during the development of cancer cachexia is inconsistent . Enhanced adipose tissue zag expression in cancer cachexia suggests that zag could be a local catabolic mediator within the tissue rather than being a biomarker of fat loss . Therefore, the ability of zag to be applied as a marker of lipid utilization in cachexia syndrome and to indirectly represent fat loss is limited . Plasma glycerol and leptin may have potential to be considered as biomarkers of lipolysis and fat mass, respectively; however, no study has defined a confirmed range and optimal cut - off points for these markers . It is not clear whether a single biomarker or combination may have the most prognostic value, as no study has assessed various combinations in a cancer population . Measuring changes in fat mass over time concurrent with circulating levels of biomarkers of fat mass would provide valuable information about application of proposed fat loss biomarkers throughout the disease trajectory . These studies would help establish valid criteria to identify loss of whole body fat mass based on changes in plasma levels of these specific biomarkers . Alterations in fat mass and composition between visceral and subcutaneous depots are divergent and vary over the cancer trajectory . For example, it remains to be determined whether decreased leptin levels indicate the loss of visceral or subcutaneous adipose tissue in cancer . Although many of the proposed biomarkers are economical, easy, and quick to quantify in plasma, further steps such as comparison of plasma levels in healthy, weight stable, and weight losing cancer patients as well as their correlation with various degrees of fat loss assessed by ct images should be considered in determining capacity for application of a prognostic biomarker of fat loss in cancer . Proper study design, combined with extensive testing, and quantitative measurement of large numbers of proteins in body fluids using advanced techniques as well as statistical validation of prognostic biomarkers are important factors in identification of fat loss biomarkers . This review confirms the need for further studies to (1) assess how alterations in fat mass is reflected in measurable biomarkers, (2) minimize variations that may confound establishment of a biomarker, and (3) increase specificity and sensitivity of methods to detect biomarkers in samples at minimum levels or in repeated measures.
A descriptive, cross - sectional design was aimed to evaluate the awareness and self - medication practices among female students in higher educational institutions using convenient sampling method for the ease of accessibility and proximity to the researchers . The study was conducted in four higher educational institutions of selangor state (one public university and three private universities), malaysia . The target sample size was 377 female students which were calculated by using raosoft sample size calculator . This sample size was calculated by keeping the population size as 20,000, power as 80%, response distribution as 50%, while confidence interval and margin of error were set at 95% and 5%, respectively . However, by considering the response rate of 80%, a total of 472 female students were contacted to participate in this study . An initial draft of the questionnaire was designed by the authors after an extensive literature review . The questionnaire was set to collect data on demographic information, the prevalence of self - medication, awareness, and practices of self - medication among the students . This questionnaire was then subjected to content validity, for which it was sent to a panel of four subject experts who then screened the questionnaire for its relevance and significance . All the experts were academicians with teaching and/or research experience in nonprescription drugs and related courses in pharmacy and medical field . The revised version was sent to a small group of 10 students for face validity who gave their opinion on making the questionnaire more simple and brief . Reliability coefficient of the questionnaire was determined by (spss, version 20 for windows, ibm corporation, armonk, new york, usa). First part assessed the demographic information such as age, course of study, ethnicity, and religion . The second section explored the information about the self - medication practices such as frequency of drug use, source of drug purchase, reasons of self - care, and common medications advised for someone else . Third segment of the questionnaire assessed the awareness of students regarding the safety of self - medication . Data were entered into (spss, version 20 for windows, ibm corporation, armonk, new york, usa). A descriptive, cross - sectional design was aimed to evaluate the awareness and self - medication practices among female students in higher educational institutions using convenient sampling method for the ease of accessibility and proximity to the researchers . The study was conducted in four higher educational institutions of selangor state (one public university and three private universities), malaysia . The target sample size was 377 female students which were calculated by using raosoft sample size calculator . This sample size was calculated by keeping the population size as 20,000, power as 80%, response distribution as 50%, while confidence interval and margin of error were set at 95% and 5%, respectively . However, by considering the response rate of 80%, a total of 472 female students were contacted to participate in this study . An initial draft of the questionnaire was designed by the authors after an extensive literature review . The questionnaire was set to collect data on demographic information, the prevalence of self - medication, awareness, and practices of self - medication among the students . This questionnaire was then subjected to content validity, for which it was sent to a panel of four subject experts who then screened the questionnaire for its relevance and significance . All the experts were academicians with teaching and/or research experience in nonprescription drugs and related courses in pharmacy and medical field . The revised version was sent to a small group of 10 students for face validity who gave their opinion on making the questionnaire more simple and brief . Reliability coefficient of the questionnaire was determined by (spss, version 20 for windows, ibm corporation, armonk, new york, usa). First part assessed the demographic information such as age, course of study, ethnicity, and religion . The second section explored the information about the self - medication practices such as frequency of drug use, source of drug purchase, reasons of self - care, and common medications advised for someone else . Third segment of the questionnaire assessed the awareness of students regarding the safety of self - medication . Data were entered into (spss, version 20 for windows, ibm corporation, armonk, new york, usa). A total of 472 questionnaires were distributed to the participants, of which 461 respondents returned the questionnaire, giving a response rate of 97.66% . As three questionnaires were incomplete, participants age varied from 16 to 29 years (mean age in years = 19.55 1.761). Majority of them were from health - related course (n = 183; 40.0%). Almost half of the participants were from chinese (n = 225; 49.1%). The prevalence of self - medication among female students in higher educational institutions was found as 57.2% (n = 262). More than one - third of the respondents (n = 146; 31.9%) practiced self - medication 23 times a year, whereas less than one - fifth of the respondents (n = 85; 18.6%) self - medicated them once every few months [table 2]. Demographic characteristics of participants frequency of taking medications without consulting a doctor the most common source of self - prescribed medicine was a pharmacy or clinics (n = 206; 45%). 21% participants (n = 96) used medications from a stock available at their home, while 8.3% (n = 38) obtained from their family or friends . Other sources of medicines included a grocery store, hospital, and supermarket [table 3]. The study also investigated the types of medication used for self - medication [table 4]. It was found that antipyretics were the most common medications used without doctor's consultation (n = 212; 89.1%). Vitamins were the second most commonly self - prescribed medicine (n = 186; 83.4%), followed by eye - drops (n = 158; 60.3%), pain killers (n = 157; 59.9%), and herbal and/or traditional supplements (n = 125; 59.9%). In addition, the study also investigated the frequency of use of each type of medication [table 4]. Vitamins were claimed to be the most frequent medicine used by female students (n = 68; 26%), while eye - drops were reported to be the second highest frequently used therapeutic category among female students (n = 28; 10.7%). As for the symptoms leading to self - medication, it was reported that fever, (n = 202; 44.1%) cough or cold (n = 195; 42.6%), and pain (n = 155; 33.8%) were the most common symptoms for self - medication [table 5]. Other common symptoms reported were menstrual pain, diarrhea, constipation, vomiting, allergy, and indigestion . Sources of medications frequencies of medicine used for self - medication symptoms leading to self - medications minor health problem was stated as the main reason for self - medication (n = 175; 38.2%). More than one - quarter of respondents was (n = 129; 28.2%) self - medicated due to previous experience and some of respondents (n = 129; 28.2%) reported to practice self - medication because of urgent medical condition . Other common reasons given were convenience, cost, time, transport, and suggestions from either a friend or a relative [table 6]. Reasons for practicing self - medication moreover, 70.1% students (n = 320) stated that they consulted friends or relatives who were not medically qualified with regard to a health - related problem [table 7], while 38.0% (n = 174) students reported that they follow the advice given by their friends or relatives . More than one - quarter students reported (n = 126; 27.5%) that they had previously advised friends and family members to take medicine . Analgesics and antipyretics (n = 79; 62.7%) were reported to be the highest recommended medicine by students to their family and friends, followed by cold and flu medicine (n = 9, 7.1%), anti - allergy drugs (n = 7; 5.6%), and cough mixture (n = 4; 3.2%) [table 8]. The common reasons reported for taking medications were previous experience (n = 102, 81.0%), problem was not severe (n = 52; 41.3%), and urgency of problem (n = 50; 39.7%) [table 9]. Awareness of respondents about the safety of self - medication was also investigated in this study [table 10]. Most respondents (n = 331; 72.3%) were well - aware about the food interactions (n = 384; 83.8%) and adverse drug reactions (n = 399; 87.1%) associated with medicines . The majority of respondents (n = 280; 61.1%) believed otc medications are as effective as prescribed medications . Frequency of consulting friends / relatives with regard to health - related problem common medications advised to someone else reasons for advising one to take medicine students awareness on the safety of self - medication the study reported the moderate prevalence of self - practice medication practices among female students . However, the prevalence of self - medication among female students is reported to be higher in malaysia elsewhere . However, it is important to consider that target sample in this study was conveniently sampled, educated students from higher educational institutions and, therefore, more than half of the reported respondents practicing self - medication might be an underestimation on the prevalence of self - medication among the community . Moreover, education could also be interpreted as a likely variation of results between the current study and previously published research . Either a pharmacy and/or clinic were reported to be the main source for obtaining medications . This is in congruence with the findings in slovenia and ethiopia where medications were mainly procured from the pharmacy . This may explain as to why pharmacy was the main source of medicine in the current study, and one can easily locate them in closely - knitted locations in urban areas . The current research reported antipyretics, vitamins, pain killers, and traditional supplements as most common therapeutic categories for self - medication . Similar findings were reported by zafar et al . In karachi, pakistan, and lucas et al . In maputa, mozambique . However, medications such as antipyretics and analgesics should be used with caution in certain groups of patients as these therapeutic categories might pose gastrointestinal problems and adverse renal effects . The current results of fever, cough, cold, and pain being the leading symptoms of self - medication are in line with the studies by zafar et al . And hassali et al . A previous study at universiti sains malaysia reported poor knowledge about the disease and its treatment, and time - saving as two major reasons for self - medication among female students . The results of current findings could be related to above - mentioned study as female students indicated minor health problems, previous experience, urgency of condition, convenience of available facilities, cost, and time - saving as reasons for practicing self - medication in this study . In recent years, national health services it is claimed that self - medication could reduce unnecessary consultation for minor ailments, consequently more time and quality of care could be spent for patients with more severe conditions . Thus, self - medication for minor illnesses could be perceived as a good practice as it is most likely to reduce the burden of malaysian healthcare professionals . Still, we endorse the views of eickhoff et al . In this regard who highlighted the role of pharmacists in detecting the drug - related problems associated with otc drugs and emphasized to enhanced pharmacist's education, training, and practice in this regard . In 2007, ministry of health and consumers association of malaysia organized a project, which was aimed to increase awareness about the rational use of medicine, and to provide consumers important information about various medications . In this study, it was surprising to discover that 38% students practice self - medication based on advised by their friends or family . Therefore, awareness on safety of self - medication is rather an important issue highlighted in this study . They would read the label that comes with medication and checked the expiration date before and after purchasing it . They were also aware that certain medications might cause adverse drug reactions and interact with food or other medication . A malaysian study reported a similar result to this study, wherein consumers knew their medications well . In this study, it was observed that female students would prefer to consult a pharmacist than a doctor . It was relatively unexpected as doctors are thought to be the main preference for medicine consultation . The results indicate that the pharmacist plays a more crucial role in self - medication practice compared to a doctor . Therefore, pharmacists should be encouraged to provide maximum pharmaceutical care to the patients . In this study however, few participants commented that doctors or pharmacists should be consulted at the first place because self - medication is risky and at times potentially harmful . On the contrary, respondents also viewed themselves not qualified enough to practice self - medication . They advocated that proper education pertaining to self - medication should be emphasized before launching the concept of self - medication . Furthermore, not only the pharmacists but also other allied healthcare professionals, physicians should also be enlightened about the wider use of over - the - counter medication when prescribing new medications . The strength of this study is that it has focused on determining the prevalence and awareness of female students about self - medication in higher education institutes of selangor, malaysia . The findings of this study would be a valuable contribution to the existing literature as not many studies have explored the topic of self - medication among female students . Furthermore, the current research confirmed the outcomes from previous studies done in developing countries, forming a strong base to design data - driven interventions to improve the practice of self - medication in malaysia . Like any research, this study draws attention to some of the unavoidable constraints of research approach that have been adopted . The study was executed in only one state of malaysia using convenient sampling techniques, and therefore, the findings cannot be generalized to all the female students in other states of malaysia . A large number of respondent were associated with healthcare - related courses which may have caused some bias in how they view self - medication . Survey bias, intentional dishonesty on the part of respondents, and recall bias may also have affected the survey results . Prevalence of self - medication was moderate among female students in the sampled higher educational institutions in selangor, malaysia . There is a need to initiate campaigns to enhance the awareness of students about self - medication practices . The role of pharmacists should be expanded to include minor illness management service in the community pharmacies.
Consumption of fish from fresh and marine bodies of waters poses an interesting paradox . On one hand, there is increasing and rather convincing observational and experimental data underscoring the beneficial effects of marine fish consumption on fetal growth and gestation [1 - 3]. On the other hand, consumption of environmentally contaminated fish has been associated with decrements in gestation and birth weight in some studies [4 - 6]. Animal evidence suggests that rhesus monkeys, rats and mice exposed to polychlorinated biphenyls (pcbs) in utero have reductions in length of gestation and growth [7 - 9]. The results from accidental environmental exposures also report adverse effects on birth size and ectodermal manifestations among fetuses to various polyhalogenated hydrocarbons . One occupational study has reported a negative relation between pcb exposure and infant birth weight, which no longer remained significant after adjusting for gestation and other variables related to birth weight . Results from a birth cohort study in the faroe islands, a community with a high intake of marine fish, whale meat and blubber, supported a relation between marine fatty acids and diminished birth weight for gestation, but the effects were not attributed to the mercury or pcb exposure . Fish consumption is a major dietary route of exposure for humans to pcbs [14 - 16]. Among anglers in the great lakes basin concern about consumption of fish from contaminated bodies of water has fostered research focusing on such consumption and pregnancy outcomes including the gestational age and birth weight of exposed infants . For example, consumption of fish from the baltic sea has been linked to preterm delivery and reduced birth weight and, possibly, fetal growth restriction . Similar associations have been reported for women consuming fish from the great lakes with an effect comparable in magnitude to that reported for cigarette smoking, i.e., 160190 grams . A few investigators have evaluated risks associated with fish consumption in the context of cigarette smoking, a known determinant of fetal growth . For example, olsen et al . Has reported a positive relation between marine fishmeals and placental weight but only among non - smoking women . Other findings remain equivocal with several authors reporting decrements in gestation and birth weight associated with fish consumption [4 - 6,20,21] while others do not [22 - 24]. To date, there has been limited attention paid to fish consumption amongst other known determinants of gestation and fetal growth in an attempt to weigh risk in relation to these factors . This gap may reflect the utilization of maternal self - report or birth certificate data for the collection of information on birth size and potential confounders . Both sources vary in terms of the validity and reliability of information on maternal pre- and gravid disease and pregnancy complications and require cautious interpretation of findings [25 - 27]. We believe that this has hampered a more complete interpretation of findings regarding the hazards of fish consumption on fetal development . The message to the public remains unclear despite the potential health hazard associated with fish consumption . This hazard reflects the persistent and lipophilic nature of pcbs and related compounds and their ability to bioaccumulate within the aquatic food chain . Consistent with this exposure scenario, we undertook a study to assess consumption of contaminated fish and birth size in the context of other determinants of gestation and fetal growth . In addition, we attempted to assess risks posed by such chronic low levels of exposure by estimating the effect of fish consumption on the proportionality of birth size . The referent study population comprised 4,226 infants born between 19861991 to 7,564 female and 10,518 male participants in the new york state angler cohort study (nysacs). This time period coincided with the five years prior to the initiation of the nysacs . Using a cross - sectional design, the nysacs selected a stratified random sample of licensed anglers aged 1840 years who resided in 16 counties in close proximity to lakes erie and ontario . Surveys were systematically mailed in four waves to 30,000 anglers to assess three research objectives in the overall nysacs: 1) characterize recent (in 1991) species specific fish consumption behaviors and knowledge of fish consumption health advisories among anglers; 2) to characterize exposure in targeted individuals to persistent environmental pollutants such as pcbs among men and women consuming large amounts of fish; and 3) to assess fish consumption in relation to reproductive and developmental endpoints . After two follow - up attempts, 39% of male and 49% of female anglers returned completed questionnaires and comprise the study cohort . In addition to completing questions regarding offspring, cohort members were individually linked to the new york state live birth registry to verify offspring using an a priori matching algorithm following institutional review board approval . The research staff at the new york state health department, who was masked to fish consumption status, confirmed all linkages . Study participants voluntarily completed an 8-page self - administered questionnaire on fish consumption habits and select sociodemographic characteristics . (questionnaire available upon request .) Given the small number of multiple births, the dependent nature of pregnancy outcomes and to minimize recall bias, we restricted the study sample to the most recent singleton births (n = 2,716; 70%). This approach recognizes the dependent nature of pregnancy outcomes (i.e., repetition of adverse outcomes such as low birth weight in successive pregnancies). Based upon post hoc power estimates, our study was able to detect a 61-gram reduction in birth weight based upon an 80% power and 5% alpha . Data were collected from two sources mailed self - administered questionnaires and hospital delivery records . The 8-page questionnaire ascertained information on lifetime fish consumption and select sociodemographic characteristics such as household income in four mutually exclusive categories . Hospital delivery records were abstracted to elicit information on known determinants of birth size and birth weight, length, head and chest circumference . Duration of fish consumption referred to the number of years consuming any fish from lake ontario, the most polluted great lake, and its tributaries . Sport fish from the great lakes fish are among the most highly exposed fish species with respect to pcbs, dioxin and mirex . However, sport fish from lakes erie and ontario are within the tolerance limits for mercury . For analysis, duration of fish consumption was categorized into four categories: none, 12, 37, and 8 + years . Women were asked to check whether they had consumed fish from lake ontario and its tributaries for each year starting with 1955 up to and including the year of birth of the index child . Cohort members were queried about frequency of fish consumption meals only for 1991 or the year they were enrolled into the nysacs . Since cohort members were selected to be between the ages of 1840 years, the period of fish consumption duration typically referred to lifetime consumption for most cohort members consistent with the persistent bioaccumulation of pcbs in aquatic ecosystems . Years consuming fish were summed into a duration variable, which has previously been found to be an important determinant of subtle alterations in human reproduction (e.g., decreased fecundability and shortened menstrual cycle length) in the nysacs . Hospital delivery records for mothers and infants were obtained for 92% of infants from 101 hospitals throughout new york state to ascertain information on known determinants of fetal growth as measured by birth size . Hospital closure or the inabilities of hospital staff to locate records were the main reasons for not obtaining missing records . Gestational age and birth size measurements were abstracted from hospital delivery records by trained nurses / researchers who were blinded to maternal fish consumption status . Health insurance and marital status were abstracted from medical records in their original formats and dichotomized for analysis insured yes / no and married / unmarried . Birth size measurements included birth weight (grams), birth length (centimeters) and head and chest circumference (centimeters). To permit comparison of birth size outcomes for infants in this study with live born infants in the united states, percentiles for birth weight, length and head circumference were established for each infant in the sample using recognized published standards . These growth standards are infant sex specific . For each infant, a ponderal index (pi) was calculated as a measure of proportionality of birth size using the established formula: 100 times birth weight in grams divided by the cube of birth length in centimeters . A second measure of proportionality and fetal growth restriction, the head to chest ratio, this ratio is derived from the following formula: head circumference in centimeters divided by chest circumference in centimeters . The pi and head to chest ratio are indices intended to give patterns of proportionality based on traditional measures of birth size, i.e., weight, length, chest circumference, and head circumference . The following study covariates or known determinants of fetal growth (34) were abstracted from hospital delivery records and operationalized in a manner most consistent with most hospital records: maternal and infant race (white / nonwhite), parity (left continuous), uterine bleeding during pregnancy (yes / no), history of previous spontaneous abortion (yes / no), cigarette smoking during pregnancy (yes / no; if yes, average number daily cigarettes smoked during pregnancy), evidence placental infarction (yes / no), infant sex (male / female), gestational age (left continuous), placental infarction (yes / no), and birth defect (yes / no). Birth defects, a heterogeneous grouping of defects recorded in hospital delivery records, were included given that affected infants are reported to have lighter or heavier birth weights depending upon the nature of the defect . In the abstraction process, nurses were instructed to note any discrepancies or uncertain information for review by the study's developmental pediatrician who made all final coding decisions while remaining blinded to fish consumption status . Descriptive statistics were performed to assess consumption patterns by select covariates and birth size measures . Statistical significance (p <0.05) was determined with the chi - square statistic or student - t test depending upon level of measurement for each variable . The analytic phase of analysis included use of analysis of variance to assess potential effect modification between fish consumption, gestational age and mean birth size measurements . Gestational age was categorized as: preterm <37 weeks, term 3741 weeks, and post - term 42 + weeks . Separate multiple linear regression models were tested to identify determinants of birth weight, length, head and chest circumference . Specification of the model for each measure of birth size included duration of maternal fish consumption in four categories, infant characteristics (i.e., gestation, sex and birth defects) and maternal characteristics (i.e., parity, placental infarction, uterine bleeding, and average number daily cigarettes smoked during pregnancy). Subsequently, the analyses were rerun after excluding 469 infants for whom birth defects were noted in hospital delivery records . Variables for each multivariate model were selected using a p = 0.25 cut point and were retained in the final model if the covariate achieved statistical significance p <0.05 (38). Duration (in four categories) of maternal sport fish consumption was forced into the model while all other covariates were entered using a forward stepwise approach . The referent study population comprised 4,226 infants born between 19861991 to 7,564 female and 10,518 male participants in the new york state angler cohort study (nysacs). This time period coincided with the five years prior to the initiation of the nysacs . Using a cross - sectional design, the nysacs selected a stratified random sample of licensed anglers aged 1840 years who resided in 16 counties in close proximity to lakes erie and ontario . Surveys were systematically mailed in four waves to 30,000 anglers to assess three research objectives in the overall nysacs: 1) characterize recent (in 1991) species specific fish consumption behaviors and knowledge of fish consumption health advisories among anglers; 2) to characterize exposure in targeted individuals to persistent environmental pollutants such as pcbs among men and women consuming large amounts of fish; and 3) to assess fish consumption in relation to reproductive and developmental endpoints . After two follow - up attempts, 39% of male and 49% of female anglers returned completed questionnaires and comprise the study cohort . In addition to completing questions regarding offspring, cohort members were individually linked to the new york state live birth registry to verify offspring using an a priori matching algorithm following institutional review board approval . The research staff at the new york state health department, who was masked to fish consumption status, confirmed all linkages . Study participants voluntarily completed an 8-page self - administered questionnaire on fish consumption habits and select sociodemographic characteristics . (questionnaire available upon request .) Given the small number of multiple births, the dependent nature of pregnancy outcomes and to minimize recall bias, we restricted the study sample to the most recent singleton births (n = 2,716; 70%). This approach recognizes the dependent nature of pregnancy outcomes (i.e., repetition of adverse outcomes such as low birth weight in successive pregnancies). Based upon post hoc power estimates, our study was able to detect a 61-gram reduction in birth weight based upon an 80% power and 5% alpha . Data were collected from two sources mailed self - administered questionnaires and hospital delivery records . The 8-page questionnaire ascertained information on lifetime fish consumption and select sociodemographic characteristics such as household income in four mutually exclusive categories . Hospital delivery records were abstracted to elicit information on known determinants of birth size and birth weight, length, head and chest circumference . Duration of fish consumption referred to the number of years consuming any fish from lake ontario, the most polluted great lake, and its tributaries . Sport fish from the great lakes fish are among the most highly exposed fish species with respect to pcbs, dioxin and mirex . However, sport fish from lakes erie and ontario are within the tolerance limits for mercury . For analysis, duration of fish consumption was categorized into four categories: none, 12, 37, and 8 + years . Women were asked to check whether they had consumed fish from lake ontario and its tributaries for each year starting with 1955 up to and including the year of birth of the index child . Cohort members were queried about frequency of fish consumption meals only for 1991 or the year they were enrolled into the nysacs . Since cohort members were selected to be between the ages of 1840 years, the period of fish consumption duration typically referred to lifetime consumption for most cohort members consistent with the persistent bioaccumulation of pcbs in aquatic ecosystems . Years consuming fish were summed into a duration variable, which has previously been found to be an important determinant of subtle alterations in human reproduction (e.g., decreased fecundability and shortened menstrual cycle length) in the nysacs . Hospital delivery records for mothers and infants were obtained for 92% of infants from 101 hospitals throughout new york state to ascertain information on known determinants of fetal growth as measured by birth size . Hospital closure or the inabilities of hospital staff to locate records were the main reasons for not obtaining missing records . Gestational age and birth size measurements were abstracted from hospital delivery records by trained nurses / researchers who were blinded to maternal fish consumption status . Health insurance and marital status were abstracted from medical records in their original formats and dichotomized for analysis insured yes birth size measurements included birth weight (grams), birth length (centimeters) and head and chest circumference (centimeters). To permit comparison of birth size outcomes for infants in this study with live born infants in the united states, percentiles for birth weight, length and head circumference were established for each infant in the sample using recognized published standards . These growth standards are infant sex specific . For each infant, a ponderal index (pi) was calculated as a measure of proportionality of birth size using the established formula: 100 times birth weight in grams divided by the cube of birth length in centimeters . A second measure of proportionality and fetal growth restriction, the head to chest ratio, was calculated for all infants . This ratio is derived from the following formula: head circumference in centimeters divided by chest circumference in centimeters . The pi and head to chest ratio are indices intended to give patterns of proportionality based on traditional measures of birth size, i.e., weight, length, chest circumference, and head circumference . The following study covariates or known determinants of fetal growth (34) were abstracted from hospital delivery records and operationalized in a manner most consistent with most hospital records: maternal and infant race (white / nonwhite), parity (left continuous), uterine bleeding during pregnancy (yes / no), history of previous spontaneous abortion (yes / no), cigarette smoking during pregnancy (yes / no; if yes, average number daily cigarettes smoked during pregnancy), evidence placental infarction (yes / no), infant sex (male / female), gestational age (left continuous), placental infarction (yes / no), birth defects, a heterogeneous grouping of defects recorded in hospital delivery records, were included given that affected infants are reported to have lighter or heavier birth weights depending upon the nature of the defect . In the abstraction process, nurses were instructed to note any discrepancies or uncertain information for review by the study's developmental pediatrician who made all final coding decisions while remaining blinded to fish consumption status . Descriptive statistics were performed to assess consumption patterns by select covariates and birth size measures . Statistical significance (p <0.05) was determined with the chi - square statistic or student - t test depending upon level of measurement for each variable . The analytic phase of analysis included use of analysis of variance to assess potential effect modification between fish consumption, gestational age and mean birth size measurements . Gestational age was categorized as: preterm <37 weeks, term 3741 weeks, and post - term 42 + weeks . Separate multiple linear regression models were tested to identify determinants of birth weight, length, head and chest circumference . Specification of the model for each measure of birth size included duration of maternal fish consumption in four categories, infant characteristics (i.e., gestation, sex and birth defects) and maternal characteristics (i.e., parity, placental infarction, uterine bleeding, and average number daily cigarettes smoked during pregnancy). Subsequently, the analyses were rerun after excluding 469 infants for whom birth defects were noted in hospital delivery records . Variables for each multivariate model were selected using a p = 0.25 cut point and were retained in the final model if the covariate achieved statistical significance p <0.05 (38). Duration (in four categories) of maternal sport fish consumption was forced into the model while all other covariates were entered using a forward stepwise approach . Infants were predominantly born to married women (93%) covered by health insurance (98%). Mothers who consumed fish were significantly older than non - consumers but the difference was negligible . Mothers consuming fish for 8 + years were more likely to be parity 2 + than non - consumers, i.e., 32% and 23%, respectively . However, no other significant differences with respect to reproductive history were observed across categories of maternal fish consumption (data not shown). Mothers in the highest fish consumption category smoked significantly more cigarettes per day than mothers in other categories of consumption . No difference in frequency or mean alcohol consumption during pregnancy was observed by consumption status . Infant and maternal characteristics of study sample by maternal fish consumption note: duration of maternal fish consumption refers to the number of years eating fish from lake ontario and its tributaries between 1955 and infant's year of birth . * p <0.05 no significant mean differences in any measure of birth size (including percentiles, ratios or indices) were observed across categories of fish consumption and gestation . One noted exception was that post - term infants whose mothers reported consuming fish for 8 + years had slightly smaller head circumferences than other infants, but these effects were negligible (<1 cm) and based on small sample sizes . Similar findings were observed for chest circumference though the findings only attained borderline (p = 0.06) significance . The results of multiple linear regression analysis where the same model was tested for each measure of birth size (excluding percentiles) are presented in table 2, and reflect the absence of a significant effect for fish consumption on birth weight, length or head circumference . Cigarette smoking during pregnancy was significantly associated with all birth size measures as were gestational age, male gender and parity . Specifically, cigarette smoking during pregnancy was associated with a predicted decrement in birth weight, length, head and chest circumference while gestation, parity and male gender conferred positive effects . Overall, the models with study covariates included accounted for between 1021% of the variation in birth size measures . Predictors of birth size multiple linear regression note: duration of maternal fish consumption refers to the number of years eating fish from lake ontario and its tributaries between 1955 and infant's year of birth . Ci = confidence interval; n = sample size for each model similar results were observed after restricting the analysis to infants without birth defects (table 3). Fish consumption was not significantly associated with birth weight, length or head circumference . However, consumption of fish for 8 + years was significantly associated with a decrement in chest circumference (-0.49; 95%ci = -0.87,-0.11). However, this finding was based on half the sample size (n = 1,050) reflecting a sizable amount of missing data on chest circumferences in hospital records . A second observed difference when restricting to infants free of birth defects was that uterine bleeding no longer remained significantly associated with birth length . However, uterine bleeding was of borderline (p = 0.0551) significance in the analysis using all infants regardless of birth defects . Predictors of birth size among infants without birth defects multiple linear regression note: restricted to infants without birth defects noted in hospital delivery records (n = 2,247). Duration of maternal fish consumption refers to the number of years eating fish from lake ontario and its tributaries between 1955 and infant's year of birth . We did not find any evidence of an adverse relation between duration of maternal consumption of fish from lake ontario and its tributaries and gestational age or birth size . Overall, our findings agree with the results of earlier investigators who reported no adverse effects associated with fish consumption [22 - 24] and support the voluminous body of literature denoting gestation, male sex, parity, cigarette smoking, placental infarction, and birth defects as determinants of fetal growth and birth size . We are using fish consumption as a proxy for pcb exposure while simultaneously assessing the effect of fish consumption on pregnancy outcomes such as gestation and birth size . Specifically, our exposure measure relies on maternal report and focuses exclusively on fish consumed from lake ontario and its tributaries . These noteworthy limitations necessitate the need for cautious interpretation in the context of previous work . Other explanations for the absence of an observed effect may be the secular decline in the contaminant load of pcbs and other contaminants in fish, residual confounding stemming from our inability to control for duration of breastfeeding prior to the index pregnancy or other unmeasured confounders . The absence of an effect for the highest categories of consumption fails to support the former explanation, which would be consistent with eating for longer periods of time including the 1970s when fish were most contaminated . Our study is unable to evaluate potential acute exposures associated with ingestion of a fish meal given that we only ascertained monthly meals for 1991 as a crude measure of recent consumption in the context of health advisories to the contrary . Upon further inspection of our data, however, we observed that the majority of women reporting having eaten fish from lake ontario and its tributaries reported eating fish in 1991 despite health advisories to the contrary . We posit that one explanation for observed differences in results across studies is the lack of attention to known determinants of gestation and fetal growth when assessing fish consumption as well as the dependent nature of pregnancy outcomes . One frequently cited study reporting a negative association with great lakes fish consumption and birth size did not appear to include cigarette smoking in the model as noted by at least one other author . A few authors have noted that the fish consumption effect, despite being negatively associated with birth size, is small in comparison to other covariates important for fetal growth and development, which is more in keeping with our findings . Several investigators have reported positive effects of maternal consumption of marine sport fish on gestation or the rate of fetal growth, or conversely an increased risk of preterm delivery or low birth weight associated with a maternal diet low in marine fish . We did not observe (nor were we able to refute) these relations, though we did observe a negative albeit small association between maternal sport fish consumption and birth size among post - term infants, which disappeared after controlling for other determinants of fetal growth as measured by birth size . Still, we do not know what aspect of fish consumption, particularly, marine fish consumption, confers the positive effect on fetal growth as reported by other investigators . The health benefits or hazards associated with fish consumption will require added attention to origin of fish marine or fresh water given the reported differences in stores of polyunsaturated fatty acids and n-3 fatty acid between the two types of fish [44 - 47]. Levels of n-3 fatty acids vary by fish species, oil content of fish, sex and age of fish, and factors exogenous to fish such as water temperature . Oily fish from cold waters (whether marine or fresh bodies of water) are reported to have higher levels of n-3 fatty acids in comparison to fish from warmer bodies . The ratio of n-3 to n-6 fatty acids has recently been suggested as being associated with gestation and birth weight and this ratio is approximately four - times greater in marine fish in comparison to fresh water fish . Type and species - specific fish consumption will remain important considerations in assessing exposure scenarios based on fish consumption . Our findings require careful interpretation for three key reasons: (1) the absence of maternal serum for quantifying pcb and other environmental exposures; (2) the retrospective reporting of fish consumption by mothers in this observational study; and (3) the potential selectiveness of our sample based on licensed fish holders who agreed to participation . Our exposure is maternally reported lake ontario fish consumption and is only a crude proxy for pcb body burden . We recognize this limitation and have focused our attention on fish consumption rather than serum pcb concentration, per se . We were unable to directly assess the validity or reliability of fish consumption for this sample of mothers with respect to life time duration of fish consumption . Rather, we have circumstantial evidence from the overall new york state angler cohort study, which attempted to evaluate the reliability (as measured by percent agreement) of self - reported fresh water fish consumption . Specifically, a sub - sample of 100 participants were randomly selected for telephone interviews approximately 36 months after receipt of the mailed self administered questionnaire . The percent agreement for the number of monthly species - specific fish meals consumed in 1991 ranged from 8589% (pearson r = 0.40.7) when comparing questionnaire and telephone interview data . With respect to response bias in the overall nysacs, non - respondents more likely to be nonwhite, to have lower educational attainment and household incomes than respondents, but did not differ with respect to reported fish consumption (unpublished data available upon request). We recognize the potential for selection bias in that only 3% to 4% of infants were preterm or low birth weight in our sample . This may reflect our use of a state fish license registry as the sampling framework for ascertaining anglers in the nysacs . As such, socially and economically disadvantaged families, traditionally at higher risk for these two adverse pregnancy outcomes, may be less able to be purchase a fish license in comparison to more advantaged families . Such sampling may have resulted in under representation of women at highest risk of having an infant born preterm or of diminished size . At this time, our findings add to rather than demystify the uncertainty regarding the relation between maternal fish consumption and infant gestation and birth size . Further clarification regarding the potential developmental sequelae associated with fish consumption remains an important public health priority, as fish remains an important dietary source of long chain n-3 fatty acids and proteins for vulnerable populations such as pregnant women, fetuses and infants . We support an earlier appeal for definitive work to demystify findings so the public can be advised accordingly (50). Such efforts should estimate risk based on valid and reliable pcb exposure data with concerted attention to other known determinants of outcomes such as gestation and fetal growth . Gmb, eff, jev, jmw, and mem were instrumental in conceptualizing the research question and study design and in preparing the paper . Pgt and ms were responsible for preparing the analytic files and implementing the analytic plan, under the direction of gmb and jmw, and in the preparation of this paper . Table comparing means for measures of birth size by maternal fish consumption and gestational age of infants, new york state angler cohort study, 1986 = 1991 supported in parts with grants from the great lakes protection fund (rm 7913021) and the agency for toxic substances & disease registry (h75/ath29832800). The authors thank bridget mcguinness for her technical assistance with file preparation, terri raimondo for programmatic technical assistance, danielle belser for verifying linkages, and mary ann snyder and sylvia vena for medical record abstraction.
Molecular profiling (mp) encompasses the testing of multiple biomarkers to evaluate the underlying genetic alterations present in a tumor at any one point in time [13]. To date molecular profiles may consist of multiple gene mutational analyses, gene copy number changes by fluorescence in situ hybridization (fish), gene expression profiles measured by microarrays (ma) and protein expression by immunohistochemistry (ihc). This approach is superior to the testing of one biomarker target which does not take into account the complexity of multiple signaling pathways and cross talk . The cancer literature is replete with studies exploring single biomarkers in clinical trials designed to test single agents or biomarker analyses performed as a post hoc analysis . Although there are a plethora of biomarkers that have emerged that may positively prognosticate or predict response to various therapies, the clinical utility and adoption of this approach has been slow due to validation concerns, reproducibility and translation into clinical care [57]. Since comprehensive mp uses a multi - dimensional approach to testing, it is inherently more complex and requires extensive validation . There is significant investment in high cost, high throughput technologies, trained laboratory work force, and laboratory informatics to achieve the level of validation required by clia or cap to offer the test for patient - care . Laboratory developed test - validations in a clia mandated environment typically requires the following: specimen type and specimen handling protocols: since this variable can hugely affect reproducibility of the test, standardization of specimen handling is imperative . More and more, the formalin fixed paraffin embedded tissue is becoming the sample of choice as it is readily available . This sample type has been shown to perform adequately for mutational analysis; gene expression profiles measured by rt - pcr or oligonucleotide arrays, fish, and ihc . However, the quality of analysis on this preferred sample type has to be closely monitored based on time to fixation, time in fixative and age of the samples . Additionally for newer molecular techniques, internal and external quality checks such as the amount or percentage of tumor nuclei, the quantity and quality of dna and rna, measurement of internal housekeeping genes are all important determinants in the overall quality of results [915]. Validation of each component of the mp assay, whether being performed in a non - profit hospital - based / academic laboratory or for - profit reference labs, has to follow the strict clia guidelines as well as guidelines provided by laboratory associations such as college of american pathologists (cap) or cls1 . For each test offered, per validation guidelines, the laboratory must document certain performance characteristics which include: accuracy to document that the test produces expected result by appropriate testing of known positive and negative samples . From these accuracy studies the analytical sensitivity, specificity and accuracy of the assay can be determinedprecision studies are performed to determine intra - run and inter - run reproducibility.the assay will also have to determine appropriate reference ranges and limit of detection for appropriate reporting of results . Ongoing quality assurance and proficiency testing are some other additional requirements by clia specimen type and specimen handling protocols: since this variable can hugely affect reproducibility of the test, standardization of specimen handling is imperative . More and more, the formalin fixed paraffin embedded tissue is becoming the sample of choice as it is readily available . This sample type has been shown to perform adequately for mutational analysis; gene expression profiles measured by rt - pcr or oligonucleotide arrays, fish, and ihc . However, the quality of analysis on this preferred sample type has to be closely monitored based on time to fixation, time in fixative and age of the samples . Additionally for newer molecular techniques, internal and external quality checks such as the amount or percentage of tumor nuclei, the quantity and quality of dna and rna, measurement of internal housekeeping genes are all important determinants in the overall quality of results [915]. Validation of each component of the mp assay, whether being performed in a non - profit hospital - based / academic laboratory or for - profit reference labs, has to follow the strict clia guidelines as well as guidelines provided by laboratory associations such as college of american pathologists (cap) or cls1 . For each test offered, per validation guidelines, the laboratory must document certain performance characteristics which include: accuracy to document that the test produces expected result by appropriate testing of known positive and negative samples . From these accuracy studies the analytical sensitivity, specificity and accuracy of the assay can be determinedprecision studies are performed to determine intra - run and inter - run reproducibility.the assay will also have to determine appropriate reference ranges and limit of detection for appropriate reporting of results . Ongoing quality assurance and proficiency testing are some other additional requirements by clia accuracy to document that the test produces expected result by appropriate testing of known positive and negative samples . From these accuracy studies the analytical sensitivity, specificity and accuracy of the assay can be determined precision studies are performed to determine intra - run and inter - run reproducibility . The assay will also have to determine appropriate reference ranges and limit of detection for appropriate reporting of results . Ongoing quality assurance and proficiency testing are some other additional requirements by clia given the resource investment requirements for conducting these multi - dimensional, labor intense assays, it is easily conceivable that these assays are increasingly being offered by large centralized laboratories . (examples include: genomic health inc ., pathwork diagnostics and caris life sciences). The rapidly developing genomic information is leading to the proliferation of mp services and assays and their subsequent introduction into clinical care . This service offers a new approach in which an evidence rated review of the literature based on the the us preventive task force rating is utilized to identify targets in tumor tissue associated with current therapies . Using a technology and platform agnostic approach, various targets are analyzed using a combination of assays such as gene sequencing, oligonucleotide microarray, mutational analyses, copy number changes using fish analysis and protein expression by ihc . This particular approach for mp to measure molecular targets was studied in a feasibility study in 2006 and most recently in a multi - center clinical trial, across nine different cancer centers in the us . Using the caris life sciences caris target now mp service, von hoff et al . Reported a longer pfs for patients on mp - directed therapy than physician choice for 27% of patients (95% ci, 1738% p = 0.007). This study used a novel study design in which the patient served as their own controls and pfs ratio was determined by actual comparison of pfs on mp therapy versus pfs on patient s last prior therapy . For the participants (18/66) who had a pfs 1.3 overall survival was 9.7 months compared to 5 months on physician directed therapy . Interestingly, mp of tumors yielded actionable targets in 98% by this assay indicating that such an approach is feasible . However, it is to be noted that the targets identified may involve off - label use of therapies . . Study was restricted to advanced stage patients with metastases and refractory tumors, the approach may have significant benefits when used earlier ., reported two patients with advanced stage colon cancers in which identification of a target mgmt by ihc with this assay, led to measurable response to temozolomide treatment with decrease in serum markers and tumor shrinkage on ct . Using the same assay, discovery of targets was also reported in a interdigitating reticulum cell sarcoma, an exceedingly rare tumor . Median time to treatment failure (ttf) in 161 patients with one aberration treated with matched targeted therapy was 5.3 months (95% ci: 4.1, 6.6) versus 3.2 months (95% ci: 2.94.0) for their prior systemic antitumor therapy (prior to referral to phase i) (p = 0.0003). For patients with one aberration, the cr - pr rate was 29% with matched targeted therapy versus 8% without matching (p = 0.0001). The cr + pr rate was 6% in 438 patients without molecular testing treated on the same studies . Interestingly, these rates compare favorably with those reported by von hoff et al . For the caris target now service . These preliminary results suggest that in early clinical trials, matching patients with targeted drugs based on their molecular profile results in (a) longer ttf compared to their prior therapy and (b) higher rates of response, survival and ttf compared to those seen in patients treated without molecular matching . The battle trial for personalizing therapy for lung cancer identified targets of high interest in treatment of lung cancer and using an adaptive randomized trial utilized real time biomarker analyses to predict sensitivity or resistance to targeted agents . A similar trial i - spy 2 also employs this groundbreaking clinical trial model that uses genetic or biological markers (biomarkers) from individual patient s tumors to screen promising new treatments, identifying which treatments are most effective in specific types of patients . In addition, this innovative adaptive trial design similar to battle trial will enable researchers to use early data from one set of patients to guide decisions about which treatments might be more useful for patients later in the trial, and eliminate ineffective treatments more quickly . With more focus on the mp of tumors and greater realization of the limitations of one - biomarker one target approach, cancer treatment in the us is about to experience a major revolution . Upfront mp of tumors at the time of diagnosis and subsequently at all points of tumor recurrence, whether local or distant, will change the treatment of oncology care forever . This will hopefully lead to better control of cancer, improved outcomes for patients, and a more rational and less expensive oncology care.
Exacerbations of copd are associated with accelerated loss of lung function, poor quality of life, and mortality.1,2 these events can be predicted by numerous clinical factors, including prior exacerbations, airflow obstruction, symptom burden, gastroesophageal reflux, and leukocytosis.3 it is important to detect copd exacerbations early and minimize their severity . Patients with copd frequently experience significant decreases in oxygen saturation during exercise, attributed to the imbalance between oxygen delivery and exercise - induced demand.4 exercise - induced oxygen desaturation (eid) is reported to be associated with hospitalization and mortality in patients with copd.5 the 6-minute walking test (6mwt) has been suggested as the preferred measure to identify patients with copd and eid.6 eid occurs frequently during the 6mwt in patients with copd.7 eid has been related to forced expiratory volume in 1 second (fev1), diffusion capacity of lung carbon monoxide (dlco), amount of emphysema, and baseline oxygen saturation.810 pulmonary hypertension (ph) is an important factor contributing to acute exacerbation of copd.11 ph appears when airflow limitation is severe, and is associated with chronic hypoxemia . Pulmonary vascular remodeling in copd is the main cause of increased pulmonary artery (pa) pressure, and is thought to result from the combined effects of hypoxia, inflammation, and capillary loss in severe emphysema.12 the presence of ph has been shown to increase the hospitalization rate and mortality of patients with copd.13,14 computed tomography (ct)-detected pa enlargement is independently associated with acute exacerbations of copd.15 the pa - to - aorta (pa: a) ratio measured by ct scan outperforms echocardiography for diagnosing resting ph in patients with severe copd.16 a pa: a> 1 indicates lower oxygen saturation at rest than a pa: a <1.15 however, there are no reports on the association between pa: a and eid in patients with copd . We hypothesized that pa: a correlates with the presence of eid and that 6mwt results are useful for predicting the risk of having a pa: a> 1 . The present study aimed to examine the relationship between pa: a and eid and develop a simple screening tool by determining the appropriate cutoff score on the 6mwt to predict a pa: a> 1 in patients with copd . This study analyzed regularly treated outpatients with copd between 2014 and 2015 at the kobe city medical center west hospital . A total of 64 patients with copd were included after applying the exclusion criteria in this study (figure 1). The criteria for diagnosing copd were a smoking history (20 pack - years) and postbronchodilator fev1/forced vital capacity (fvc) <70% . Furthermore, we used the following inclusion criteria to define copd clinically, all of which had to be fulfilled: symptoms, including cough, sputum production, wheezing, dyspnea, smoking history (20 pack - years), existence of emphysema on chest ct, and a physician diagnosis of copd.1721 study - exclusion criteria were as follows: history of lung surgical procedures, exacerbation - related hospitalization 3 months before 6mwt, and patients on long - term oxygen therapy . This examination included an assessment of body weight, height, and medical history (eg, pulmonary embolism and sleep apnea syndrome), gold (global initiative for chronic obstructive lung disease) grade 04, history of acute exacerbations of copd within the previous year, copd assessment test, level of dyspnea (using the modified medical research council dyspnea scale), postbronchodilator spirometry, dlco, 6mwt (according to international recommendations), emphysema area, and pa enlargement on ct . Body mass index (bmi) was calculated as weight in kilograms divided by height in meters squared . Gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80%, symptoms, including cough, sputum production, wheezing, and dyspnea, smoking history (20 pack - years), existence of emphysema on chest ct, and a physician diagnosis of copd.1721 the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat, round, 25 m walking course supervised by a physician and physical therapist . A pulse oximeter (wristox 3150; nonin medical, plymouth, mn, usa) with a finger probe measured peripheral oxygen saturation (spo2) during 6mwt, and 6mwt - analysis software (wristox 2; star product, tokyo, japan) was used . In addition, a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt . Eid was defined as a nadir spo2 <90%, spo2 88%, and spo2 4%.2325 one reviewer, blinded to hemodynamic information, analyzed ct scans (optima ct 660 discovery; ge healthcare, little chalfont, uk). Measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa: a ratio, as previously reported.1416 in cases where the aorta was not uniform in diameter, two measurements were taken 90 apart and the larger diameter used . Pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa: a> 1 (figure 2).1416 results are expressed as counts or median (interquartile range). Cohen s -coefficient measured intraobserver and interobserver agreements for ct measurements of the pa: a ratio . Bivariate analyses were performed with the use of pearson s test for categorical data and the mann spearman s rank - correlation coefficient was determined for relationships between the pa: a ratio, lung - function parameters, 6mwt parameters, and ct parameters . Receiver operating characteristic (roc) curves were used to determine the threshold values with the best sensitivity and specificity to predict pa: a> 1, with the best being defined as the point on the roc curve with the shortest distance from the upper - left corner . Sensitivity, specificity, positive / negative predictive value, and positive / negative likelihood were calculated for lung - function parameters and 6mwt parameters of exacerbation - risk factors on the basis of a previous study.6,27,28 all statistical analyses were performed with ezr (saitama medical center, jichi medical university, saitama, japan), which is a graphical user interface for the r project (r foundation for statistical computing, vienna, austria).29 more precisely, it is a modified version of r commander designed to add statistical functions frequently used in biostatistics, and p - values <0.05 were considered statistically significant . This study analyzed regularly treated outpatients with copd between 2014 and 2015 at the kobe city medical center west hospital . A total of 64 patients with copd were included after applying the exclusion criteria in this study (figure 1). The criteria for diagnosing copd were a smoking history (20 pack - years) and postbronchodilator fev1/forced vital capacity (fvc) <70% . Furthermore, we used the following inclusion criteria to define copd clinically, all of which had to be fulfilled: symptoms, including cough, sputum production, wheezing, dyspnea, smoking history (20 pack - years), existence of emphysema on chest ct, and a physician diagnosis of copd.1721 study - exclusion criteria were as follows: history of lung surgical procedures, exacerbation - related hospitalization 3 months before 6mwt, and patients on long - term oxygen therapy . This examination included an assessment of body weight, height, and medical history (eg, pulmonary embolism and sleep apnea syndrome), gold (global initiative for chronic obstructive lung disease) grade 04, history of acute exacerbations of copd within the previous year, copd assessment test, level of dyspnea (using the modified medical research council dyspnea scale), postbronchodilator spirometry, dlco, 6mwt (according to international recommendations), emphysema area, and pa enlargement on ct . Body mass index (bmi) was calculated as weight in kilograms divided by height in meters squared . Gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80%, symptoms, including cough, sputum production, wheezing, and dyspnea, smoking history (20 pack - years), existence of emphysema on chest ct, and a physician diagnosis of copd.1721 the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat, round, 25 m walking course supervised by a physician and physical therapist . Subjects were encouraged using standard methodology every minute of the 6mwt . A pulse oximeter (wristox 3150; nonin medical, plymouth, mn, usa) with a finger probe measured peripheral oxygen saturation (spo2) during 6mwt, and 6mwt - analysis software (wristox 2; star product, tokyo, japan) was used . In addition, a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt . 90%, spo2 88%, and spo2 4%.2325 one reviewer, blinded to hemodynamic information, analyzed ct scans (optima ct 660 discovery; ge healthcare, little chalfont, uk). Measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa: a ratio, as previously reported.1416 in cases where the aorta was not uniform in diameter, two measurements were taken 90 apart and the larger diameter used . Pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa: a> 1 (figure 2).1416 this examination included an assessment of body weight, height, and medical history (eg, pulmonary embolism and sleep apnea syndrome), gold (global initiative for chronic obstructive lung disease) grade 04, history of acute exacerbations of copd within the previous year, copd assessment test, level of dyspnea (using the modified medical research council dyspnea scale), postbronchodilator spirometry, dlco, 6mwt (according to international recommendations), emphysema area, and pa enlargement on ct . Body mass index (bmi) was calculated as weight in kilograms divided by height in meters squared . Gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80%, symptoms, including cough, sputum production, wheezing, and dyspnea, smoking history (20 pack - years), existence of emphysema on chest ct, and a physician diagnosis of copd.1721 the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat, round, 25 m walking course supervised by a physician and physical therapist . A pulse oximeter (wristox 3150; nonin medical, plymouth, mn, usa) with a finger probe measured peripheral oxygen saturation (spo2) during 6mwt, and 6mwt - analysis software (wristox 2; star product, tokyo, japan) was used . In addition, a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt . One reviewer, blinded to hemodynamic information, analyzed ct scans (optima ct 660 discovery; ge healthcare, little chalfont, uk). Measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa: a ratio, as previously reported.1416 in cases where the aorta was not uniform in diameter, two measurements were taken 90 apart and the larger diameter used . Pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa: a> 1 (figure 2).1416 cohen s -coefficient measured intraobserver and interobserver agreements for ct measurements of the pa: a ratio . Bivariate analyses were performed with the use of pearson s test for categorical data and the mann spearman s rank - correlation coefficient was determined for relationships between the pa: a ratio, lung - function parameters, 6mwt parameters, and ct parameters . Receiver operating characteristic (roc) curves were used to determine the threshold values with the best sensitivity and specificity to predict pa: a> 1, with the best being defined as the point on the roc curve with the shortest distance from the upper - left corner . Sensitivity, specificity, positive / negative predictive value, and positive / negative likelihood were calculated for lung - function parameters and 6mwt parameters of exacerbation - risk factors on the basis of a previous study.6,27,28 all statistical analyses were performed with ezr (saitama medical center, jichi medical university, saitama, japan), which is a graphical user interface for the r project (r foundation for statistical computing, vienna, austria).29 more precisely, it is a modified version of r commander designed to add statistical functions frequently used in biostatistics, and p - values <0.05 were considered statistically significant . The current analysis comprised 64 patients who were separated into groups on the basis of pa: a> 1 (n=29) and 1 (n=35). The -values for intraobserver and interobserver agreements for detecting pa: a> 1 were 0.87 (95% confidence interval [ci] 0.740.99) and 0.75 (95% ci 0.580.91), respectively . Differences in the pa: a ratio between both groups were driven by the diameter of pa (2.9 [2.73.3] cm in pa: a 1 vs 3.7 [3.53.9] cm in pa: a> 1, p=0.002), because no differences were detected in the diameter of aortae (3.7 [3.43.9] cm vs 3.5 [3.33.7] cm, p=0.20). There were no significant differences between the two groups with regard to age, sex, bmi, pack - years, modified medical research council dyspnea scale, gold, copd assessment test, baseline pulse rate, or baseline modified borg scale (p>0.05). On the other hand, there were significant differences between the two groups with regard to fev1 (71.6% [60.5%80.8%] vs 52.6% [39.6%72.1%], p=0.013), fvc (82.3% [50.3%93.6%] vs 75.8% [42.7%86%], p=0.04), fev1:fvc ratio (68% [61%73.3%] vs 53.8% [48.8%69.4%], p=0.023), dlco (72.5% [55.5%82.9%] vs 44.6% [37.7%49.6%], p=0.005), bode (bmi, obstruction [airflow], dyspnea, and exercise performance) index (2 [13] vs 4 [25], p<0.001), 6mw distance (6mwd; 450 m [400510.5] vs 325 m [238466], p<0.001), baseline spo2 (97% [95%97.5%] vs 95% [93%96%], p=0.001), lowest spo2 (92% [91%94%] vs 86% [84%88%], p<0.001), highest modified borg scale result (2 [05] vs 5 [25], p=0.04), low - attenuation area (laa; 6.8% [2.8%14.7%] vs 25.4% [11.3%33.4%], p<0.001), and history of acute exacerbations of copd within the previous year (1 [2.9%] vs 7 [24.1%], p=0.019) (table 1). The pa: a ratio demonstrated a significant linear correlation with lowest spo2 (r=0.68, r=0.46; p<0.001), dlco (r=0.61, r=0.37; p<0.001), 6mwd (r=0.43, r=0.18; p<0.001), bode index (r=0.41, r=0.17; p<0.001), baseline spo2 (r=0.36, r=0.13; p=0.003), laa (r=0.36, r=0.13; p=0.004), fvc (r=0.34, r=0.12; p=0.006), fev1 (r=0.29, r=0.08; p=0.019), and highest pulse rate (r=0.26, r=0.07; p=0.035) (table 2). Using roc curves, the threshold values with the best cutoff point, sensitivity, and specificity to predict pa: a> 1 were determined for spo2 during the 6mwt (best cutoff point 89%, area under curve [auc] 0.94, 95% ci 0.881), dlco (best cutoff point 51%, auc 0.87, 95% ci 0.780.96), 6mwd (best cutoff point 388 m, auc 0.75, 95% ci 0.620.87), and bode index (best cutoff points 4, auc 0.74, 95% ci 0.610.87) (table 3, figure 3). The performance data on the 6mwt and lung function for predicting pa enlargement are depicted in table 4 . Spo2 <90%, spo2 88%, and spo2 4% during 6mwt were 94.3 (80.899.3), 97.1 (85.199.9), and 45.7 (28.863.4), respectively, for specificity, 93.1 (77.299.2), 95.8 (78.999.9), and 59.6 (44.373.6), respectively, for positive predictive value, and 16.2 (4.262.8), 27.7 (4193.3), and 1.8 (1.32.4), respectively, for positive likelihood ratios . First, we found a strong association between the pa: a ratio and lowest spo2 during the 6mwt . For this reason, a consistent finding in patients with copd is the close relationship between severity of hypoxemia and pa pressure or pulmonary vascular resistance, supporting a major role for alveolar hypoxia.30 alveolar hypoxia causes constriction of the resistance pas, and sustained alveolar hypoxia induces pulmonary vascular remodeling.28 pathological studies of lung specimens from patients with copd have shown extensive pulmonary vascular remodeling, with prominent intimal thickening and medial hypertrophy . For this reason, chronic alveolar hypoxia plays an important role in pulmonary vascular remodeling.28 in a previous study, patients with pa: a> 1 showed lower resting spo2, higher usage rates of supplemental oxygen, and an indirect association with eid.15 in the present study, the lowest spo2 during the 6mwt to predict pa: a> 1 was considered to be a beneficial result based on the roc curves . These results suggest that the lowest spo2 during the 6mwt is a very helpful measure and screening test for the pa: a ratio . For example, it might be possible to easily screen for pulmonary artery expansion by means of the six - minute walking test in a home - care situation, where it would be difficult to perform ct imaging . Second, with regard to the relationship between pa: a ratio and lung function, correlations were observed among fev1, dlco, and laa . One of the factors that may play a role in causing ph to advance in patients with copd is the destruction of lung parenchyma, leading to loss of part of the pulmonary vascular bed,30,31 and the burden of airway remodeling influencing pa - pressure increase.28 a previous study included patients with airflow - obstruction severity greater than moderate, and our study included mild airflow obstruction and smokers with normal spirometry.15 therefore, regardless of the severity of airflow obstruction, pa enlargement may be progressing . Undiagnosed copd is a problem worldwide.18 gold 0 has been reported to be an exacerbation risk; therefore,18 early detection and not just spirometry evaluation is important from multiple perspectives.18,32 from the viewpoint of early detection of pa enlargement, a definition of eid as spo2 <90% may be a good start . However, these findings may imply that pa: a> 1 is one of the multiple risk factors for acute copd exacerbations . One reason for this is that pa: a is associated with ph16 and ph is also a risk factor for acute copd exacerbations.33 furthermore, a previous study reported an association between the pa: a ratio and acute copd exacerbation.15 these results suggest that screening for the pa: a ratio without ct using the 6mwt may indicate the risk of acute copd exacerbations at an early stage . This study had some limitations, including small size, single - center experience, and retrospective design . In addition, this study also included copd subjects who did not fit the gold criteria . Furthermore, because healthy controls do not have respiratory symptoms and there are no control data for the measurement items pertaining to such individuals, healthy controls were not included in the present study . However, it has been reported that the presence of clinical symptoms and low dlco in smokers, even with normal spirometry, increases the risk of progression to airflow obstruction in copd.1720 therefore, the present study s results during the 6mwt could be useful to screen for ph at an early copd stage, even in gold 0 patients . Finally, according to a previous study, left ventricular dysfunction causes pa enlargement . However, echocardiography was not performed in all subjects, and this information could not be included because it was unavailable from the medical history, although we observed clinically relevant associations between ct - measured pa: a ratios and 6mwt results . The current study s findings suggest that there is a strong association between pa: a ratio and lowest spo2 during the 6mwt . Lowest spo2 during the 6mwt is a very helpful measurement to predict pa: a ratios on ct, and lowest spo2 of <89% during the 6mwt is excellent to screen for ph in copd.
It is an infectious disease caused by the parasite plasmodia (p.) transmitted by the female anopheles . Four identified species of this parasite exist, which cause different types of human malaria . Although all the four species of malaria parasites can infect humans and cause illness, only p. falciparum is known to be potentially life threatening and some of infected persons die, usually because of delayed treatment; however, annual incidence of clinically new cases and mortality rates are decreasing [36]. As malaria vaccines remain problematic, chemotherapy still is the most important weapon in the fight against the disease . The antimalarial drugs including chloroquine, quinine, mefloquine, pyrimethamine, and artemisinin are currently used in malaria treatment . Part of the reason for the failure to control malaria is the spread of resistance to first - line antimalarial drugs, cross - resistance between the limited number of drug families available, and some multidrug resistance . Resistance has emerged to all classes of antimalarial drugs except artemisinin, an endoperoxide antimalarial drug derived as the active component of artemisia annua, a herbal remedy used in chinese folk medicine for 2000 years qinghaosu artemisinin is a natural product and a powerful antimalarial drug with significant activities, which has high potency whilst possessing low toxicity during treatment of malaria [1315]. The genus artemisia has always been of great pharmaceutical interest and is useful in traditional medicines for a treatment of the variety of diseases [11, 16, 17]. A. annua is presently being cultivated on a commercial scale in china and vietnam for its antimalarial sesquiterpene lactone . The genus is of small herbs found in northern temperate regions and belongs to the important family compositae (asteraceae), which comprises about 1,000 genera and over 20,000 species . Within this family, artemisia is included into the family anthemideae and comprises itself over 400 species, found in europe and north america, but mainly are dominating asia [1820]. Among the asian artemisia flora, 150 species were recorded for china, 50 species reported in japan, and 34 species found in iran, of which may be endemic: a. melanolepis boiss and a. kermanensis pold, a. absinthium, a. annua, a. dracunculus, a. aucheri, a. haussknechtii boiss, a. scoparia, a. sieberi, and a. sieberi besser . Pharmacochemical analysis of artemisinin shows that the structure of this compound is rather unique among natural products as it contains the very unusual 1,2,4-trioxane ring system . It was sufficiently unusual that it was originally characterized as an ozonide until revised crystallographic analysis provided unambiguous structural elucidation [2933]. For a drug to be effective against the malaria parasite, it must reach the site of action in sufficient concentration and then interact with the receptors before it is either deactivated and/or eliminated by the host or the parasite . Pharmacological and biochemical evaluation revealed that this compound was a blood schizonticide, preferentially imported into malaria infected erythrocytes via the parasitophorous duct as it has been also shown in noninfectious diseases . Due to complex chemical structure of artemisinin, the chemical synthesis of the molecule is complex, which results in very low yields, and the cost becomes prohibitory to use synthetic approach for its commercial production . The mechanism of the action of artemisinin remains a mystery; several candidates have been hypothesized as targets of artemisinin, including iron, haem, and some parasite membrane proteins [3739], pfatp6; however, none of these has been convincingly shown to be functionally relevant and need to be debated . Pharmacochemistry and chemical analysis of different genus of iranian artemisia species has been studied, and the presence of variety of components including monoterpenes, sesquiterpenes [43, 44], sesquiterpene lactones [45, 46], and essential oils [4751] was fully reported [2228]. The aim of this study is pharmacochemistry of natural components of iranian flora artemisia sieberi and its antimalarial effects on plasmodium berghei in vivo . This is the first application of a. sieberi for treatment of murine malaria so far . The aerial parts of a. sieberi were collected at flowering stage from the khorassan and semnan provinces of iran . Voucher specimens were deposited and identified at the herbarium of the research institute of forests and rangelands (rifr), tehran, iran . The powder (140 gr) of a. sieberi was macerated in 1 lit methanol (merck) and then kept for 72 h away from light and high temperature . It was filtered, evaporated, and dried by rotary evaporator (eyela, n-1000, japan) and finally defatted in refrigerator . Wet weight of raw extract at the final step was 13.3 gr, and its color was dark green . Herbal extract was eluted with 300 ml n - hexane (sigma, co. india); two phases were separated; the lower hexane phase (non - polar compounds) was collected and kept at refrigerator for further experiment . The upper phase was eluted with 300 ml chloroform (merck, india) 3 times; subsequently lower chloroform phase was collected, evaporated, and extracted . Higher methanol phase was then eluted with 300 ml diethyl ether (merck, india) 3 times . It is suggested that semi polar components could be separated in these two chloroform and ether phases . Male out bred nmri (naval medical research institute) mice (supplied by the laboratory animal department, karaj production and research complex, pasteur institute of iran) were used in this study . The mice were 46 weeks of age and almost 20 g weight which were housed at room temperature (2023c) on a 12 h light and 12 h dark cycle, with unlimited access to food and tap water . Experiments with animals were done according to the ethical standards formulated in the declaration of helsinki, and measures taken to protect animals from pain or discomfort . It has been approved by institutional ethical review board (ethical committee of the pasteur institute of iran), in which the antimalarial test was done . (a) toxicity assay of a. sieberi herbal extract in nave animals in vivo toxicity was assessed by using herbal extract on nave nmri male mice . Animals were divided into four groups (n = 5 mice / group), including group 1 (nave), group 2 (low dose), group 3 (average dose), and group 4 (high dose). According to several publications of this laboratory [5154], in a blind experiment with no previous findings, three different concentrations ranging from 1 and 100 mg / ml can be used . A sample of herbal extract was suspended in ethanol and normal saline (1: 9), then three different concentrations (low, average, and high doses) of herbal extracts including 1, 10, and 100 mg / ml were tested in vivo for their toxicity as test animals and a control group which was injected with drug vehicle . Entire animals in all groups were injected with 200 l of related solutions subcutaneously (sc) once a day for 5 days . In vivo toxicity animals were divided into four groups (n = 5 mice / group), including group 1 (nave), group 2 (low dose), group 3 (average dose), and group 4 (high dose). According to several publications of this laboratory [5154], in a blind experiment with no previous findings, three different concentrations ranging from 1 and 100 mg / ml can be used . A sample of herbal extract was suspended in ethanol and normal saline (1: 9), then three different concentrations (low, average, and high doses) of herbal extracts including 1, 10, and 100 mg / ml were tested in vivo for their toxicity as test animals and a control group which was injected with drug vehicle . Entire animals in all groups were injected with 200 l of related solutions subcutaneously (sc) once a day for 5 days . (b) antimalarial effects of herbal extract on p. berghei infected micefollowing toxicity assay, the highest dose with the lowest toxicity of herbal extract (100 mg / ml concentration) was selected to apply for its antimalarial activity on male nmri mice infected with p. berghei . Animals were divided into two groups (n = 10 mice / group), including control and test; both groups were infected with murine malaria parasite, p. berghei . Drug vehicle and herbal extract were injected sc into control and test groups, respectively, once a day with 200 l of solutions for the period of 10 days . Following toxicity assay, the highest dose with the lowest toxicity of herbal extract (100 mg / ml concentration) was selected to apply for its antimalarial activity on male nmri mice infected with p. berghei . Animals were divided into two groups (n = 10 mice / group), including control and test; both groups were infected with murine malaria parasite, p. berghei . Drug vehicle and herbal extract were injected sc into control and test groups, respectively, once a day with 200 l of solutions for the period of 10 days . (c) antimalarial effects of ether and chloroform extracts on p. berghei infected micethe antimalarial efficacy of ether and chloroform extracts was investigated on murine malaria p. berghei infected nmri mice . Animals were divided into four groups (n = 5 mice / group), including ether extract control and test, chloroform extract control and test groups . Drug vehicle and extracts were injected sc into control and test groups, respectively, once a day with 200 l of solutions for the period of 14 days . The antimalarial efficacy of ether and chloroform extracts was investigated on murine malaria p. berghei infected nmri mice . Animals were divided into four groups (n = 5 mice / group), including ether extract control and test, chloroform extract control and test groups . Drug vehicle and extracts were injected sc into control and test groups, respectively, once a day with 200 l of solutions for the period of 14 days . The significance of differences was determined by analysis of variances (anova) and student's t - test using graph pad prism software (graph pad, san diego, ca, usa). Results of this experiment were classified in the following three steps including (a), (b), and (c). (a) toxicity assay of a. sieberi herbal extract in nave animalsno toxicity was observed in vivo even with high dose of a. sieberi total extract . Pathophysiological signs including body weight, survival rate, hepatomegaly, and splenomegaly represented no side effects of total extract (figure 1). No toxicity was observed in vivo even with high dose of a. sieberi total extract . Pathophysiological signs including body weight, survival rate, hepatomegaly, and splenomegaly represented no side effects of total extract (figure 1). (b) antimalarial effects of total herbal extract on p. berghei infected miceno side effects on pathophysiology were represented by total extract in malarial mice (figure 2). The results indicated significant effects of total extract on reducing parasitaemia in test group in comparison with control group (figure 3). No side effects on pathophysiology were represented by total extract in malarial mice (figure 2). The results indicated significant effects of total extract on reducing parasitaemia in test group in comparison with control group (figure 3). (c) antimalarial effects of ether and chloroform extracts in p. berghei infected micelow pathophysiological changes including survival rate and hepatomegaly were indicated after treatment in test groups when compared with those in control groups (figure 4). The results indicated the inhibitory effects of the a. sieberi ether and chloroform extracts on malaria by high reduction degree of parasitaemia (figure 5). Low pathophysiological changes including survival rate and hepatomegaly were indicated after treatment in test groups when compared with those in control groups (figure 4). The results indicated the inhibitory effects of the a. sieberi ether and chloroform extracts on malaria by high reduction degree of parasitaemia (figure 5). Although various species of the genus artemisia were used for their pharmacological, antimicrobial, and antioxidant activity, only few species of this genus including a. scoparia, a. sieberi, and a. aucheri are widely distributed in desert area of iran . This investigation is the first report on application of a. sieberi extracts on the treatment of murine malaria . The results of this assessment showed no toxicity even with high concentration of herbal extract, which confirms its minimal side effects . In spite of less efficacy of crude extract of herb, ether and chloroform extracts although a significant reduction was observed in the percentage of parasitaemia, no pathophysiological alterations were indicated in host hepato / splenomegaly and in body weight . The microscopic examination of giemsa - stained slides showed a virtual absence of blood stage of the murine malaria treated with this herbal extracts . These observations suggest that the active constituents in the extract may be cytotoxic for p. berghei, thereby inhibiting their development to the erythrocytic stage . In authors' previous publications [5256], antimalarial effects of different iranian flora of artemisia herbal extracts including a. turanica a. khorassanica, a. diffusa, a. absinthium, and their effective agent (tehranolide) against malaria and/or leishmania were successfully evaluated . Although subcutaneous injection was used in this study, other routes may be recommended for future studies . In addition to authors' previous publications [5256], data of this study specifically indicated the inhibitory effects of the a. sieberi ether and chloroform extracts on the developmental stages of p. berghei by decreasing parasitaemia . The microscopic examination of giemsa - stained slides showed a virtual absence of blood stage of the murine malaria treated with these herbal extracts . These observations suggest that the active constituents in the extract may be cytotoxic for p. berghei, thereby inhibiting their development to the erythrocytic stage . Although this study confirmed antimalarial effects of a. sieberi extracts against murine malaria in vivo during infection; however, there are more efficacies on pathophysiological symptoms by this medication . These observations provide the basis for the traditional use of this herb in treatments of malaria disease . Conclusively, some efficacies are indicated on reducing pathophysiological symptoms by this medication . However, there is requirement to find the major component of this herbal extract by further studies . More investigations are required on different plasmodia and animal hosts to clarify details of antimalarial effects of a. sieberi and analysis of its natural components.
Multiple myeloma (mm) is one of the most common hematological malignancies, the majority of which remain incurable . The common presenting symptoms of mm, such as bone pain, asthenia, fatigue, weight loss, and dyspnea, are nonspecific, which may contribute to the significant delay in the diagnosis in almost half of mm cases after the onset of the first symptom; furthermore, 20% of patients have no or mild symptoms at the time of diagnosis . There might be other possible less well - known clinical features associated with mm; for example, eczema may slightly increase the risk of the igg myeloma subtype . We report a case where eczema preceded the diagnosis of mm by at least 5 years in the hope of adding confirmatory evidence for the association between eczema and myeloma . With the advancements in the therapeutic approaches to myeloma, the recognition of early myeloma symptoms would lead to prompt diagnosis and treatment before the development of serious organ damage, and prolong life . A 68-year - old male presented for progressive loss of appetite and lethargy for 3 weeks . The past medical history included hypertension, hyperlipidemia, coronary artery disease with multiple percutaneous coronary interventions (the most recent one was 3 weeks prior to the admission), congestive heart failure with automated implantable cardioverter - defibrillator placement, and psoriasis diagnosed 5 years ago . The patient had a history of smoking one pack of cigarettes per day for 40 years . There were multiple large erythematous plaques of skin in the bilateral inguinal and axillary and right hip areas weeping serous discharge (fig . The laboratory tests showed normal hemoglobin initially, which dropped to 8.5 within 1 month after admission, hypercalcemia, m spike, and elevated 2-microalbumin and igg levels (table 1). The ct scan of the head demonstrated multiple punched out lucencies within the skull (fig . Bone survey revealed innumerable lytic lesions throughout the visualized bones: cranial vault (fig . 2b), proximal shaft of femur bilaterally, hips, and pelvic bones (fig . 2c, d). Bone marrow core biopsy showed increased plasma cells up to 10%, confirmed on cd138, with predominance of monoclonal lambda . Skin biopsy revealed epidermal spongiotic dermatitis with acanthosis and parakeratosis, the upper dermis with perivascular lymphocytic exocytosis and rare eosinophils . In addition, the patient had eosinophilia and elevated ige (table 1); atopic eczema was the most likely diagnosis . Mm, one of the most common hematological malignancies, represents 1.4% of all new cancer cases in the united states and accounts for 2% of cancer - related mortality . Although the 5-year survival has been improving, most of the mm cases remain incurable . In addition, there has been a rise in the incidence of new mm cases of 0.7% each year over the last decade . The revised criteria for the diagnosis of mm issued by the international myeloma working group (imwg) utilize biopsy and clinical clues (table 2). The evidence supporting the diagnosis of mm in this case includes: (1) 10% clonal plasma cells on bone marrow biopsy (see case description); (2) corrected calcium above 11 mg / dl; (3) although the renal function was normal at admission, the creatinine level reached 3.1 during the hospital course transiently; (4) the hemoglobin fell to 8.5 g / dl after admission (table 1), and (5) extensive lytic bone lesions on ct and bone survey (fig . Although not required by the revised imwg diagnostic criteria, the presence of an m spike provides further support of the diagnosis of mm (table 1). The levels of 2-microalbumin (3.7 mg / l) and albumin (2.7 g / dl) help classify the mm as stage ii igg lambda mm, according to the international staging system for multiple myeloma (table 3). The common presenting symptoms of mm are nonspecific, i.e. Bone pain, asthenia, fatigue, weight loss, and dyspnea . About 20% of patients have no symptoms or mild ones at the time of diagnosis . More than 40% of mm patients were diagnosed more than 6 months after the onset of the first symptom . The prolonged delay commonly occurs in patients who first present to their general practitioner and is associated with more complications, advanced stage of malignancy, and reduced disease - free survival . Therefore, it is critical to increase the awareness of myeloma and associated symptoms among medical communities, especially general practitioners . We report a case of mm where eczema occurred 5 years prior to the diagnosis of mm . Unexplained eczema of adult onset has been reported to be a marker of a variety of hematologic malignancies and solid tumors [6, 7, 8]. The risk of mm is positively related to the number and duration of inflammatory conditions; a prior history of eczema has been reported to slightly increase the risk of myeloma . Therefore, in the absence of an identifiable etiology, recalcitrant eczema in an adult should raise the suspicion of an internal malignancy, including mm . The possibility of mm should be evaluated systematically with meticulous physical examination, complete blood count, blood film, serum protein electrophoresis, chest x - ray, and ct scan of the trunk . The revised criteria for the diagnosis of mm issued by the international myeloma working group (imwg) utilize biopsy and clinical clues (table 2). The evidence supporting the diagnosis of mm in this case includes: (1) 10% clonal plasma cells on bone marrow biopsy (see case description); (2) corrected calcium above 11 mg / dl; (3) although the renal function was normal at admission, the creatinine level reached 3.1 during the hospital course transiently; (4) the hemoglobin fell to 8.5 g / dl after admission (table 1), and (5) extensive lytic bone lesions on ct and bone survey (fig . Although not required by the revised imwg diagnostic criteria, the presence of an m spike provides further support of the diagnosis of mm (table 1). The levels of 2-microalbumin (3.7 mg / l) and albumin (2.7 g / dl) help classify the mm as stage ii igg lambda mm, according to the international staging system for multiple myeloma (table 3). The common presenting symptoms of mm are nonspecific, i.e. Bone pain, asthenia, fatigue, weight loss, and dyspnea . About 20% of patients have no symptoms or mild ones at the time of diagnosis . More than 40% of mm patients were diagnosed more than 6 months after the onset of the first symptom . The prolonged delay commonly occurs in patients who first present to their general practitioner and is associated with more complications, advanced stage of malignancy, and reduced disease - free survival . Therefore, it is critical to increase the awareness of myeloma and associated symptoms among medical communities, especially general practitioners . We report a case of mm where eczema occurred 5 years prior to the diagnosis of mm . Unexplained eczema of adult onset has been reported to be a marker of a variety of hematologic malignancies and solid tumors [6, 7, 8]. The risk of mm is positively related to the number and duration of inflammatory conditions; a prior history of eczema has been reported to slightly increase the risk of myeloma . Therefore, in the absence of an identifiable etiology, recalcitrant eczema in an adult should raise the suspicion of an internal malignancy, including mm . The possibility of mm should be evaluated systematically with meticulous physical examination, complete blood count, blood film, serum protein electrophoresis, chest x - ray, and ct scan of the trunk.
An increased incidence of hashimoto thyroiditis has been reported in patients with turner syndrome . In view of its pathogenic relationship with autoimmune thyroiditis we report the case of a 9-yr - old girl with turner syndrome who developed graves disease during recombinant human gh therapy, and we discuss previous reports of this association . The patient was the first child of healthy unrelated parents of normal stature, father 175.0 cm and mother 159.0 cm . The patient was born after an uncomplicated pregnancy at a gestational age of 37 wks and 1 d. her birth weight was 2490 g. on examination, lymphedema and webbed neck were detected . Chromosomal analysis of peripheral blood lymphocytes (n=26) showed complete deletion of the short arm of the second x chromosome (karyotype: 45, x). At the age of 5 yr and 2 mo, her height was 98.2 cm (2.25 sd) and her weight was 14.4 kg (1.48 sd). A gh stimulation test was performed, and the peak gh values after stimulation with clonidine and arginine were 8.39 ng / ml and 5.56 ng / ml, respectively . Of note is that diagnosis of complete gh deficiency is less than 5 ng / ml in this assay . Recombinant human (rh) gh therapy (0.175 mg / kg / wk) was initiated with success (fig . Thyroid function tests showed a tsh level of 0.01 u / ml (normal, 0.50 to 5.50 u / ml), a free (f) t4 value of 6.00 ng / dl (normal, 0.85 to 1.80 ng / dl), and a free t3 value of 21.76 pg / ml (normal, 2.50 to 5.50 pg / ml). Anti - thyroglobulin antibody was 3.4 u / ml (normal, <0.3 u / ml). Anti - thyroid peroxidase antibody was 5.5 u / ml (normal, <0.3 u / ml). Tsh receptor antibody (trab) was 52% (normal, <15%). These results are compatible with a diagnosis of graves disease and treatment with thiamazole was started . Growth curve of the patient at the latest follow up (9 y old), the development of the patient s breast was tanner stage 1 . Treatment with thiamazole was continued and the thyroid function has stayed within normal ranges . The association of turner syndrome with graves disease was reported in english about 1020 years ago (1,2,3,4,5,6,7,8,9,10). Table 1table 1 graves disease in turner syndrome summarizes the historical thirteen known cases of graves disease with turner syndrome, including our case . This table reveals that the clinical characteristics of graves disease with turner syndrome are similar to those known generally in patients without turner syndrome in terms of ages of the onset, symptoms and prognosis . Treatment with rhgh was given to 4 patients in table 1, and it was continued in 3 out of the 4 patients including our case . This continuation of gh treatment, together with the successful treatment of hyperthyroidism, suggests that a direct link between gh treatment and graves disease is unlikely . Consistent with this hypothesis, the incidence of hyperthyroidism in japanese patients with turner syndrome receiving rhgh therapy is 0.40%, which is similar to overall incidence of hyperthyroidism in patients with turner syndrome, 00.5% (11, 12). It is well known that hyperthyroidism in patients with turner syndrome leads to acceleration of height velocity (6, 13). In our patient, the height velocity was not increased by hyperthyroidism, probably because her growth had been already accelerated by rhgh therapy before the onset of hyperthyroidism . It is important to monitor changes of thyroid function as well as growth parameters in patients with turner syndrome, as is recommended by sybert and mccauley (14).
The laboratory mouse has emerged as the major mammalian model for studying human genetic and multi - factorial diseases . Numerous mouse mutants have been produced and, more recently, technological improvements have allowed mouse mutants for virtually any gene to be produced by gene - specific approaches (knock - outs, knock - ins and conditional mutagenesis). Random approaches such as large scale, genome - wide enu mutagenesis and gene trapping have also expanded the current repertoire of available mutants . Using these mouse mutants, researchers are able to decipher molecular disease and potentially develop new diagnostic, prognostic and therapeutic approaches . The international knockout mouse consortium [ikmc (http://www.knockoutmouse.org); (1,2)] is made up of four major projects (eucomm (http://www.eucomm.org) in europe, komp (http://www.nih.gov/science/models/mouse/knockout/) and tigm (http://www.tigm.org) in the usa and norcomm (http://www.norcomm.org) in canada, and is in the process of producing mutations in es cells for all known protein coding genes . In particular, some 650 mouse lines are being produced and phenotyped in high - throughput screens as part of the eucomm and eumodic projects (http://www.eumodic.org), the results of which will be presented in the europhenome resource (3). To take this process to the next level, the international mouse phenotyping consortium (impc) has recently been formed with a remit to raise the funding for and to coordinate the production of mouse mutants for each of the ikmc mutations, along with high throughput phenotyping of these mice resulting in the first complete catalogue of mammalian gene function (see appendix 6 of the prime final report: http://www.prime-eu.org/prime final report.pdf). Archiving and distribution of the products of these various projects is a vital activity, alongside the capture of data describing in detail the genotype and phenotype characteristics of the mutants . The costs for a typical academic researcher to regenerate from scratch one of these knock - out (ko) lines has been estimated at 2530 k and would take at least 9 months . Regenerating the mouse lines is an obvious waste of public funds for science as well as laboratory mice from an animal welfare aspect . Since no single archiving facility can retain all of these mutant mouse strains it is essential that all mutants that have been created are held in centrally organised repositories, from which mutant mice can readily be made available to interested investigators (4,5). The european mouse mutant archive [(emma); (6)] is a leading international network infrastructure for archiving and provision of mouse mutant strains for the whole of europe and worldwide . To provide the best possible service to the international scientific community there is a requirement for coordination of archiving and distribution of the valuable genetically defined mice and es cells in line with global research demand . The federation of international mouse resources [(fimre); (7)], of which emma is a founding member and the european component, was initiated in response to this need for coordination . As well as coordination of archiving, there is a requirement for a common portal that allows searching of all publicly available mice, including those not from fimre partners, followed by redirection to individual repositories for more detailed information and the possibility to order material . The international mouse strain resource [imsr (http://www.findmice.org); (8)] has been developed to fulfill this need and over the last few years, emma has become one of the largest mouse network repositories worldwide and a major contributor to imsr . Emma also has a special role in the archiving and distribution of mouse mutants as it is one of four repositories handling the mouse resources produced by the ikmc initiative (emma archiving and distributing the mutant mice arising out of the eucomm project, the komp repository (http://www.komp.org) handling komp products, the canadian mouse mutant repository [cmmr (http://www.cmmr.ca); (9)] handling the norcomm resources and tigm handling its own products . Eventually, these four resources will provide access to data and material covering the complete, functional characterised, proteome of the mouse, providing an unprecedented resource for bench scientists studying all aspects of the mammalian genome including human disease . The emma resource database described in this paper provides up to date information about the archiving status of mice and describes the genetic and phenotypic properties of all the mutant strains that emma stocks . The emma database has two main benefits to the research community: (i) scientists with particular gene or genes of interest can discover if any mouse lines exist with mutations in these gene(s) and what the observed phenotype changes were, which may provide clues to the gene's role, and (ii) it allows scientists to order existing mouse mutants for further research and generation of data of interest to other researchers . As well as providing user - friendly searching and browsing of the database, the emma website is the link to the scientific community and facilitates the submission of mice to the emma and requests of mice from emma, as well as expressing interest in strains currently undergoing archiving . The data recorded for each strain is a combination of data entered by the original submitting scientist as well as subsequent curation to correct and add extra value to the database . Although the full record is only available through the emma database, summary data is exchanged with our partners in ikmc and the imsr to ensure that researchers using the portals available at their sites see descriptions of emma lines, along with links back to the original record in emma and the option to order biological material . In addition, emma utilises the biomart data management system (10,11) and the distributed annotation system [das; (12)] to allow distributed, integrated querying with other resources such as the ensembl genome browser (13). The emma website is used to advertise the goals of the project and encourage interested parties to submit mouse mutant lines of widespread use to the scientific research community as a disease model or other research tool . The submission process is handled automatically by the website and collects extensive data through a web form and stores this directly in the emma database . Data collected at this stage includes: contact details for the strain producer.strain name, affected gene(s) and mutant allele(s).genetic background of the original mutation and current background.genetic and phenotype descriptions of the line.bibliographic data on the line.whether the mouse models a human disease and an omim i d if appropriate.whether the strain is immunocompromised.whether homozygous mice are viable and fertile and if homozygous mating are required . Additional optional data collected includes: affected chromosome, dominance pattern and es cell line(s) used for targeted mutants.name and description for chromosome anomaly lines.mutagen used for induced mutant lines.promoter, founder line number and plasmid / construct name(s) for transgenic lines.breeding history of the line.current health status of the line and specific information for animal husbandry such as diet used.how to characterise the line by genotyping, phenotyping or other methods e.g. Coat colour.research areas the mouse is useful for, and whether it is a research tool such as a cre - recombinase expressing line . Whether the strain is immunocompromised . Whether homozygous mice are viable and fertile and if homozygous mating are required . Current health status of the line and specific information for animal husbandry such as diet used . How to characterise the line by genotyping, phenotyping or other methods e.g. Coat colour . Research areas the mouse is useful for, and whether it is a research tool such as a cre - recombinase expressing line . Extensive curation takes place to correct and augment the initial submission data . To facilitate input of correct data by submitting users, specific tools have been incorporated into the submission form, for searching and selecting approved gene, allele, background names, symbols and identifiers (from the mouse genome database (mgd) developed by the mouse genome informatics (mgi; http://www.informatics.jax.org) group (14). Similar tools for searching and selecting pubmed bibliographic references and identifiers have also been implemented . However, there is still a requirement for manual correction of submitted data using our curation interfaces . The curation is based on the application of international rules and standards for the initial assignment and periodic review and update of the strain and mutation nomenclature, as defined by the international committee on standardized genetic nomenclature for mice (http://www.informatics.jax.org/mgihome/nomen). These approved definitions make use of control vocabularies for gene, allele, background names and symbols . Specific automated routines and associated manual curation procedures have been defined and implemented, in particular, for: assigning to each submitted strain record a unique emma identification (i d) as the primary attribute for internal strain identification and retrieval and cross - reference with connected databases such as imsr.checking that the submitted records of mutant genes or expressed transgenes (and corresponding alleles), carried by the deposited strains, have assigned the correct names, symbols and identifiers, and mutation classification (as defined by mgi) according to the associated bibliographic references.proposing new mutant gene and allele names, symbols and identifiers for publication in the mgd database, according to the associated bibliographic references or personal communication with submitting scientists.checking that the submitted backgrounds of deposited strains have approved names and symbols assigned.inserting a preliminary strain designation for each newly submitted strain, including the assigned strain background name and the mgi allele symbol, and associating it with the corresponding emma strain id.reviewing and approving the preliminary strain designations, in collaboration with the curation group at imsr.periodically reviewing and updating of current strain designations, according to variations of mgi gene and allele's names and symbols.automated correction and population of bibliographic data using the submitted pubmed ids and the citexplore web service (http://www.ebi.ac.uk/citexplore/). Assigning to each submitted strain record a unique emma identification (i d) as the primary attribute for internal strain identification and retrieval and cross - reference with connected databases such as imsr . Checking that the submitted records of mutant genes or expressed transgenes (and corresponding alleles), carried by the deposited strains, have assigned the correct names, symbols and identifiers, and mutation classification (as defined by mgi) according to the associated bibliographic references . Proposing new mutant gene and allele names, symbols and identifiers for publication in the mgd database, according to the associated bibliographic references or personal communication with submitting scientists . Checking that the submitted backgrounds of deposited strains have approved names and symbols assigned . Inserting a preliminary strain designation for each newly submitted strain, including the assigned strain background name and the mgi allele symbol, and associating it with the corresponding emma strain i d . Reviewing and approving the preliminary strain designations, in collaboration with the curation group at imsr . Periodically reviewing and updating of current strain designations, according to variations of mgi gene and allele's names and symbols . Automated correction and population of bibliographic data using the submitted pubmed ids and the citexplore web service (http://www.ebi.ac.uk/citexplore/). Archiving of submitted mice is handled by one of the emma mouse archiving partners (cnr instituto di biologia cellulare in monterotondo, italy; the cnrs centre de distribution de typage et darchivage animale in orleans, france; the mrc mammalian genetics unit in harwell, uk; the karolinska institute in stockholm, sweden; the helmholtz zentrum mnchen in munich, germany; the wellcome trust sanger institute in hinxton; the institut clinique de la souris in strasbourg and the cnb - csic, centro nacional de biotecnologia in madrid). The archiving process involves genotype and/or phenotype verification of the mouse, followed by test freezing of either sperm or embryos and then checking the stock can be reconstituted from this frozen stock . Several strains are in particularly high demand as they represent extremely interesting disease models or valuable cre - expressing lines and these are kept as live stocks facilitating a fast delivery to the customers . The emma lines are supplied to the research community for research purposes only and there is no charge for the cryopreservation service . Archiving of mice produced by the eucomm mouse production centres follows the same procedure except the initial import of data describing these lines is automated from the eucomm database . The emma database is used internally by the emma partners to track each mutant strain through the archiving process . For example, the status of the strain in the archiving pipeline, which centre is archiving the strain, the funding source for this archiving, which material is currently in stock and available to order is all stored in the database . Emma archiving centres record this data using internal interfaces implemented using java spring and hibernate technologies . Requests for emma mice are also submitted via the emma website and recorded in the emma database . The archiving centres again track the whole process of distributing the requested mice using the database and the same internal java interfaces . Emma now contains over 1700 submitted strains from 19 countries including around 50 lines from the usa, canada and australia . In the coming 5 years, it is predicted that there will be a tripling of the mouse lines held, largely as a result of the ikmc initiative . Although nearly 58% of the requests for mutant mouse lines were from european scientists, about one - third come from the usa and canada and requests from asia are steadily increasing . So far, emma has shipped mice to scientists from more than 500 different institutions located in 39 countries . Considering the estimated cost of generating these lines from scratch the existence of the emma resource has saved the worldwide community 37 m and 934 years of laboratory effort . The emma database can be searched using a user - friendly query interface (figure 1). This search takes full / partial case - insensitive terms and searches against the official mgi gene symbols e.g. Otog, the official imsr designated strain name e.g. B6.129s2-otog / orl, the common strain name e.g. Otogc57bl/6j, the phenotype description e.g. Auditory functions or emma ids e.g. Em:01820 . Emma lines are also browsable by the affected gene, mutant type (e.g. Targeted knock - out, targeted knock - in), particular research tools (e.g. Cre - expressing lines) or mice produced by large projects (e.g. Eucomm lines). Results of searches or browsing are presented in a table, sortable by any of the columns, which lists the emma i d, gene affected (with hyperlinks back to mgi pages describing the particular gene and mutant alleles in detail), common strain name, approved international name and a link to either order the line or express interest in ordering lines that are in the process of being archived . The latter option triggers an automated process, in which the particular archiving centre is informed that there is a priority for this line and when it becomes available further automated emails inform the original scientist that they can go ahead and complete the ordering process relevant strains can be identified by either (i) typing case - insensitive, full / partial terms in the top text field which searches against the affected gene symbols and name, approved international designated and common strain names, phenotype description and emma i d, or (ii) browsing through a complete list of lines or partial lists categorised by the affected gene(s), mutant type (targeted, gene trap, transgenic, induced, chromosomal anomalies or spontaneous), research tool [cre recombinase expressing strains, lines for tetracycline (tet)-regulated gene expression systems], strains provided by the wellcome trust knockout mouse resource and finally strains produced out of the eucomm programme . Results are presented as a table of the emma i d, affected gene, common and approved international strain names alongside links to order or register interest in ordering a line when it becomes available . Clicking on any of the common strain names pops up a description of the strain . Browsing and searching for mouse lines in emma . Relevant strains can be identified by either (i) typing case - insensitive, full / partial terms in the top text field which searches against the affected gene symbols and name, approved international designated and common strain names, phenotype description and emma i d, or (ii) browsing through a complete list of lines or partial lists categorised by the affected gene(s), mutant type (targeted, gene trap, transgenic, induced, chromosomal anomalies or spontaneous), research tool [cre recombinase expressing strains, lines for tetracycline (tet)-regulated gene expression systems], strains provided by the wellcome trust knockout mouse resource and finally strains produced out of the eucomm programme . Results are presented as a table of the emma i d, affected gene, common and approved international strain names alongside links to order or register interest in ordering a line when it becomes available . Clicking on any of the common strain names pops up a description of the strain . Clicking on any of the strain names pops up a strain description (figure 2) including the mutation type, genetic background it is currently maintained on, genetic and phenotype descriptions if known, the original producer, literature references, the genotyping or phenotyping protocol needed to confirm the mutation, what material is available along with delivery times and costs and a link for downloading associated material transfer agreement (mta) documentation, if applicable . Data presented includes the mutation type, genetic background it is currently maintained on, brief genetic and phenotype descriptions if known, the original producer, literature references, the genotyping protocol needed to confirm the mutation, what material is available along with delivery times and costs and a link for downloading associated mta documentation, if applicable . Emma strain descriptions . Data presented includes the mutation type, genetic background it is currently maintained on, brief genetic and phenotype descriptions if known, the original producer, literature references, the genotyping protocol needed to confirm the mutation, what material is available along with delivery times and costs and a link for downloading associated mta documentation, if applicable . As described earlier, a subset of data on each of the emma curated lines are sent weekly to the imsr, allowing users searching this common catalogue of mutant lines to be redirected to our site for more detailed data and the ability to order the line . The mgd database provides extensive descriptions of known mutant alleles and emma links to the mgd pages, wherever possible as the definitive source for this data . As well as our simple search box, we also provide an advanced biomart query interface, which is linked from the main search page (figure 3). The biomart interface queries a denormalised snapshot of the emma database that is updated nightly . Queries can involve complex combinations of query terms including the affected gene symbols and mgi ids, common and official strain names, emma ids, mutant type, original and maintained genetic backgrounds and the type of material available (frozen embryos, sperm or ovaries, live mice on shelf or mice rederived from frozen stock). The results are fully configurable, allowing any combination of the fields presented in the standard emma search results and strain descriptions to be displayed, as well as extra data such as whether the mutant is viable and fertile when homozygous and whether it is required to keep it homozygous, whether the line is immunocompromised, if it represents a human model, the breeding history and for targeted mutants known dominance and es cell line used, and for transgenics the promoter and plasmid construct used . The results can be previewed and exported in a number of formats such as html, tab / comma - separated text or excel . However, the real benefit of biomart comes from the ability to perform integrated querying with biomarts deployed on other resources, which share a common identifier such as mgi or ensembl ids . For example, in figure 3a biomart query has identified all lines held in emma that have an affected gene annotated by ensembl as being located on the first 100 mbp of chromosome 1 and having a transmembrane protein domain . This interface allows advanced querying of the emma database as well as distributed and integrated querying with the ensembl resource . In this example emma targeted knock - out lines are identified that have affected genes annotated by ensembl as being located within the first 100 mbp of chromosome 1 and containing a transmembrane domain in their protein products . The results table is fully configurable from within the interface and here shows the strain name, emma i d (hyperlinked back to the strain description at emma), gene symbol and phenotype description from the emma biomart and the ensembl gene i d, chromosome, start and end from the ensembl biomart located at the wellcome trust sanger institute, hinxton, uk . This interface allows advanced querying of the emma database as well as distributed and integrated querying with the ensembl resource . In this example emma targeted knock - out lines are identified that have affected genes annotated by ensembl as being located within the first 100 mbp of chromosome 1 and containing a transmembrane domain in their protein products . The results table is fully configurable from within the interface and here shows the strain name, emma i d (hyperlinked back to the strain description at emma), gene symbol and phenotype description from the emma biomart and the ensembl gene i d, chromosome, start and end from the ensembl biomart located at the wellcome trust sanger institute, hinxton, uk . A new portal is currently being developed for the ikmc initiative by the international - data coordination center (i - dcc; http://www.i-dcc.org). This will be released late 2009 and will display the status of all genes in the mutagenesis pipeline along with available products and data for the mutant es cells and mouse lines . The portal will utilise a number of biomarts developed for the ikmc component mutagenesis pipelines and repositories, as well as for other useful resources such as the gxd (15) and eurexpress (http://www.eurexpress.org) gene expression databases, and the europhenome phenotyping resource . The emma biomart will form an integral component of this ikmc portal and in addition allow a wider variety of integrated queries from our emma biomart server . Another type of data integration is provided by our distributed annotation system (das) server (www.emmanet.org/das). This serves up summary level data for each emma line, allowing the display of emma strains on das clients such as the ensembl genome browser . For example by browsing to http://www.ensembl.org/mus_musculus/gene/externaldata/emma?g=ensmusg00000055694 and clicking on the configure this page option and selecting emma it is possible to view any emma lines that exist for this gene (gdf1). The emma i d, affected gene symbol, name and link to curated data at mgi is given along with the mutation type, phenotype summary and a link to the strain description at emma . The number of mutant mouse lines submitted to emma as well as the number of requests for these mutants is likely to increase significantly in the near future . This is due to the large scale and systematic efforts of the ikmc to perform saturation mutagenesis of the mouse genome using gene targeting and gene trapping approaches . As well as continuing to expand the number of lines curated and distributed by the emma resource, collaboration with international efforts to present all available mutants worldwide is going to become ever critical as the ikmc and eventually the impc initiatives continue to produce and characterise mutants . Data exchange with imsr will continue to provide a common access site and emma will collaborate extensively with the i - dcc to provide a central portal to the data and products produced by the ikmc . There will be a particular focus on utilising the phenotyping data arising out of these programmes to allow searching for mouse models using precise phenotype queries structured using the mammalian phenotype (mp) ontology (16). The emma project is currently funded until 2013, but obviously long term, stable funding for the data storage and mouse archiving that emma performs will be critical to capture and maintain the products emerging from the ikmc and impc programmes . This is a recognised issue and the european commission is currently funding a number of projects under the esfri roadmap with the goal of identifying sources of long term funding for key scientific activities . Infrafrontier (http://www.infrafrontier.eu) is one of these projects and is tasked with securing such funding for archiving and phenotyping of mouse mutants . Infrafrontier has already decided that the archiving aspect will be taken care of by a major upgrade to the emma project . Hence, it is highly likely that emma will continue providing this valuable service to the worldwide scientific community for many years to come.
A 28-year - old healthy male was seen in our clinic complaining of vision loss in his right eye (od). Best - corrected visual acuity (bcva) was od: 20/200 and left eye (os): 20/20 . Mild vascular abnormalities were detected in the temporal aspect of right optic nerve, but no conclusive information was obtained . Oct using the stratus oct 3 model (carl zeiss meditec, jena, germany) was indicated for the evaluation of the macular areas, rnfl thicknesses, and optic nerves . The fast macular protocol revealed subtle macular thickness beyond normal in the superior and nasal quadrants of both maculae . No visible alterations in the internal microarchitecture of the retina were observed using several retinal lines and optic disc protocols . A slight incremental thickening of the rnfl was observed in the superior and nasal quadrants of the os . In order to evaluate the patient's visual field (vf) and retinal sensitivities, a frequency doubling technology perimetry (fdt), using the commercially available (matrix) device, was performed in both eyes . The threshold 30 - 2 strategy revealed the presence of junctional scotoma composed by a central scotoma in od associated with superior temporal quadrantanopia in the fellow eye . The pattern detected in the vf suggested the presence of an expansive mass at the level of the optic chiasm [fig . 1]. Magnetic resonance imaging (mri) was performed which disclosed the presence of a tumor, compatible with the diagnosis of pituitary adenoma [fig . 2]. After a complete neurological evaluation, medical treatment with cabergoline (a dopamine agonist), after 8 months, a significant reduction of tumor volume was achieved . Moreover, the patient experienced a full recovery of bcva and vf [fig . 3]. Right: superior temporal quadrantanopia in os central nervous system mri . Left: pituitary dependent macroadenoma . Visual loss associated with a junctional scotoma is a well - known clinical sign related with the presence of a compressive mass in the chiasmal area . Monteiro described a generalized reduction of the rnfl thickness in the retinas of patients with band atrophy secondary to pituitary tumors . Lederer and colleagues reported on the usefulness of the macular map thickness, while studying patients with glaucomatous optic nerve alterations . Due to a relative incremental change in the ganglion cells axonal concentration within the macular area interestingly, the patient described here showed a thickening in the nasal and superior and nasal aspects of both maculae . This tomographic sign, not visible on the biomicroscopic fundus examination and in the fa, was interpreted as an early retinal manifestation produced by the presence of the tumor . The physiopathology of this localized, incremental thickness is merely speculative, but could be produced by an alteration in the axonal transport in ganglion cells . After obstruction of the axoplasmic flow, an early enlargement (intracellular edema) of the nerve fibers followed by a chronic atrophy could occur . Though no direct evidence exists corroborating this phenomenon, at least three indirect clinical issues support this theory: (1) the retinal thickness increment was revealed anatomically by the oct, but no evidence of dye leakage was seen in the fa . (2) the presence of a topographic correlation between the junction scotoma and the macular thickening . (3) the visual loss was ipsilateral to the main tumor location . Furthermore, the macular thickness enlargement was symmetric while the visual alteration was roughly asymmetric . This most likely could be indicative of the potential for intracellular edema preceding the visual function alteration and this thickening could be an early sign that could be detected before consolidated fiber atrophy occurs . Frequency doubling technology perimetry (matrix fdt) has shown to be a suitable method for studying and detecting abnormalities in patients with neurological visual field deficits . During the functional evaluation of this patient, the fdt revealed the presence of a junctional scotoma compatible with the tumor location . Even with an asymmetric vf defect, the oct was able to show symmetric involvement, with respect to macular thickness . We speculate that the anatomical alteration revealed by the oct precedes the functional deficit found in the vf . Thus, we propose that detection of bi - superonasal macular edema (me) could be an early sign of chiasmal pathology . With an early diagnosis and subsequent treatment, a complete restoration of visual function was achieved in this case, stressing the importance of early recognition of the disease . We are unaware of previous reports regarding localized bi - superonasal me secondary to a pituitary adenoma and could find no references to it in the medical literature . Studies including a cohort of patients are warranted for the evaluation of these tomographic findings . In conclusion, it is important to suspect pituitary adenoma in cases of vf deficit and oct macular alterations.
When patients present with acute occlusion of either the internal carotid artery (ica) or the proximal middle cerebral artery (mca) within the therapeutic time window for restoration of cerebral blood flow, rapid recanalization using intravenous / intra - arterial (ia) thrombolysis and ia mechanical embolectomy is the goal of acute stroke management.11) however, if the patient presents beyond the therapeutic time window, life - threatening brain swelling and herniation, known as malignant mca infarction, can manifest within one week after the onset of stroke symptoms, along with a further decrease of consciousness and pupillary dilatation, necessitating early application of a decompressive hemicraniectomy for achievement of a better clinical outcome.24) the present article reviews the pathophysiology, historical background in previous studies, operative timing, surgical technique and clinical outcomes of decompressive hemicraniectomy for malignant mca infarction involving the mca territory with or without anterior cerebral artery and posterior cerebral artery territories . Acute occlusion of either the ica or the proximal mca and insufficient collateral blood flow result in cerebral infarction of the mca territory and associated severe brain edema . Cytotoxic edema results from failure of sodium - potassium adenosine triphosphatase in brain cell membranes and is followed by vasogenic edema in association with disruption of the blood - brain barrier . The space - occupying lesion inside the cranial vault increases the intracranial pressure, thereby reducing the cerebral perfusion pressure and cerebral blood flow . In particular, a space - occupying lesion in a unilateral hemisphere causes pressure gradients between the supratentorial and infratentorial compartments and between the bilateral supratentorial compartments . The transtentorial uncal herniation and the resultant displacement of the subthalamic - upper brainstem structures will impair the consciousness of the patient . Thus, the aim of decompressive surgery is external herniation of the swollen infarcted brain for relief of brainstem compression and to reduce intracranial pressure . The original purpose of decompressive hemicraniectomy was to help patients survive in cases of acute large hemispheric infarction.25) various case reports, retrospective studies and trials have suggested that this surgical treatment lowers mortality without increasing the incidence of severely disabled survivors.8)13)15)23)27)29) three european randomized, controlled clinical trials were conducted between 2001 and 2007.10)12)32) however, the decimal (decompressive craniectomy in malignant middle cerebral artery infarction) trial in france and destiny (decompressive surgery for the treatment of malignant infarction of the middle cerebral artery) trial in germany were stopped due to slow recruitment of cases and significant difference in mortality between groups.12)32) notwithstanding, the hamlet (hemicraniectomy after middle cerebral artery infarction with life - threatening edema trial) study, conducted in the netherlands, was completed and published in 2009.10) thirty two patients were randomly assigned to undergo surgical decompression and another 32 patients received the best medical treatment over a five - year period . According to the results, the use of hemicraniectomy resulted in a reduction in the number of case fatalities and poor outcomes (modified rankin scale score 5) for patients with large hemispheric infarction who were treated within 48 hours of stroke onset . The clinical outcome for patients is considered to show improvement with early surgical decompression before or immediately after any neurological deterioration related to brain swelling.10)22)26)28)31)33)36) this clinical deterioration includes pupil asymmetry, an altered consciousness level and aggravated hemiplegia.6) patients commonly manifest such neurological deterioration within one week after the onset of stroke symptoms . In particular, one third of patients show deterioration within 24 hours, while another third show deterioration 24 - 48 hours after symptom onset.24) to date, many clinical and radiological data defining early predictors of malignant hemispheric infarction have been reported.6)7)9)14)16)18)20)30)34) malignant edema after acute infarction can be predicted based on the volume of the infarcted brain tissue, however, extension of the initial infarct territory, delayed spontaneous recanalization of the occluded vessel, hemorrhagic transformation of the infarcted brain and the fluid volume state of the patient can all make the prediction difficult . Thus, the predictors lack a sufficient predictive value with regard to selection of candidates for a decompressive hemicraniectomy prior to neurological deterioration . In addition, in order to ensure timely decompressive surgery, all patients with acute large hemispheric infarction should be observed in an intensive care unit or stroke unit setting . Thus, determination of strict cutoff criteria with a high specificity and positive predictive value for malignant infarction is needed.21) the predictive value of the infarct volume assessed by early ct scan after stroke onset has not been found to be satisfactory, as follows: (1) hypodensity covering> 50% of mca territory within five hours after symptom onset was predictive of a malignant course with a sensitivity of 61% and specificity of 94%;34) (2) hypodensity covering> 50% of mca territory within 12 hours was predictive with a sensitivity of 64% and specificity of 66%;17) (3) hypodensity covering> 50% and 67% of mca territory within 18 hours was predictive with a sensitivity of 58% and 45%, respectively and specificity of 94% and 100%, respectively.7) one useful predictor is the initial infarct volume assessed using diffusion - weighted magnetic resonance (mr) imaging> 145 cm within 14 hours after acute mca occlusion, which achieved a sensitivity of 100% and a specificity of 94% in the study by oppenheim et al.20) the cutoff criteria for lesion volume and associated midline brain shift for prediction of malignant edema should differ according to the timing after stroke onset.4)21) gerriets et al . Reported a midline shift 2.5, 3.5, 4.0 and 5.0 mm in transcranial color - coded duplex sonography as a predictor of malignant edema after 16, 24, 32 and 40 hours, respectively, after stroke onset with a specificity of 100% and positive predictive value of 100%.4) meanwhile, assessment of the final infarct volume can be performed using perfusion ct or perfusion mr images on admission, however, such perfusion parameters are still not optimal.3)30) in the mr perfusion study reported by thomalla et al ., perfusion lesion volume> 162 ml on a time - to - peak (ttp) map with a ttp delay threshold of> 4 seconds was found to predict malignant infarction with a sensitivity of 83% and specificity of 75%.30) conceptually, decompressive craniectomy procedures include both internal and external decompression . In the case of external decompression, the frontal, temporal and parietal bones overlying the infarcted hemisphere are removed, allowing for external herniation of the swollen infarcted brain . With the patient in a state of general anesthesia, a skin incision is started just above the zygomatic arch 0.5 cm anterior to the tragus and then carried superiorly and posteriorly over the ear and around the parietal bone to the contralateral frontal midpupillary line . The hemicraniectomy then involves the removal of a large fronto - temporo - parietal bone flap, as large as possible . The bone flap is made anteriorly in order to avoid violation of the frontal sinus, except in the case of a huge frontal sinus . The medial limit is 2 cm from the midline, in order to minimize venous bleeding on the dura . The posterior limit of the bone flap is approximately 5 - 6 cm posterior to the external auditory canal, thereby covering the mca territory posteriorly and allowing for a neutral head position without compressing the brain . Inferiorly, the temporal squama is removed to the level of the zygomatic arch . Following a stellate - shaped dural incision, it is commonly recommended that the infarcted brain tissue not be removed due to the presence of a salvageable penumbra area or viable tissue.10)12)32) however, the internal decompression (removal of the infracted brain tissue and/or an anterior temporal lobectomy) can be performed for patients with whole hemispheric infarction.19) expansive duraplasty is then performed using a large flap of pericranial tissue or an artificial dura substitute . The dimensions of the expansive duraplasty should be extended in order to accommodate the subsequent aggravation of the brain edema . Multiple dural tenting sutures, bipolar coagulation of bleeding points on the dural surface, application of commercial hemostatic materials and placement of a closed suction drain in the epidural / subgaleal space are all used . Finally, the temporalis muscle and skin flap are reapproximated and sutured layer by layer . However, the temporalis muscle and fascia can be resected in order to maximize external herniation of the swollen brain . Removal of the temporalis muscle does not cause problems with chewing, as the grinding phase of the closure stroke only requires one - third of the maximal bite force and only leads to a minimal decrease in the maximal bite force.1)5) resection of the temporalis muscle and fascia provides a two - fold volume expansion on average when compared with the conventional technique on postoperative day 3 (fig . 1).22) technical obstacles to obtaining the best external decompression are an insufficient craniectomy size, epidural / subgaleal hematoma, thick and swollen temporalis muscle, tough and inelastic temporalis fascia and tight scalp.2)22)35) making hemicraniectomy as large as possible, meticulous hemostasis and resection of the temporalis muscle and fascia will all maximize external herniation of the infarcted brain . Cranioplasty is then performed using the autogenous bone flap two to three months after the craniectomy . After decompressive surgery for malignant mca infarction, clinical outcomes at 6 - 12 months after stroke onset have been reported . For the criteria determining favorable and unfavorable outcomes, a dichotomization of the mrs score between 0 - 3 and 4 to death or between 0 - 4 and 5 to death has been used; however, an mrs score of 0 - 3 is most appropriate for a favorable outcome as it includes independent functional outcomes . In the hamlet study, the patients were randomly assigned to undergo surgical decompression or to receive the best medical treatment within four days of stroke onset.10) the decompressive - surgery group had a lower incidence of mortality (22% versus 59%) and mrs 5 - 6 (41% versus 59%) than the group that received the best medical treatment . However, the incidence of patients with mrs of 0 - 3 did not differ between the two groups (25% versus 25%). Results of a pooled analysis of the three european randomized controlled trials indicated a better clinical outcome than that of the hamlet study.31) performance of decompressive surgery within 48 hours of stroke onset resulted in reduced mortality and an increase in the number of patients who had a favorable functional outcome . In addition, more patients in the decompressive - surgery group had mrs 0 - 3 (43% versus 21%) and mrs 0 - 4 (75% versus 24%) compared with those in the control group who received the best medical treatment . In analysis of the three predefined subgroups of the decimal trial, in the decompressive - surgery group, younger age showed correlation with a favorable outcome and there was a trend toward a worse outcome in patients with higher infarct volumes.32) however, no significant difference in the clinical outcome with mrs scores was observed between surviving patients with dominant and non - dominant hemisphere infarction . In patients with malignant hemispheric infarction, decompressive surgery can reduce the number of cases of fatality and increase the number of favorable outcomes . In particular, if a decompressive hemicraniectomy is performed early, before irreversible cerebral herniation, using appropriate surgical techniques, favorable outcomes with functional independence can be achieved in a high proportion of patients.
Six species of echinococcus have been recognized, but four of them are of public health concern (1). The greatest prevalence of hydatidosis in human and animal hosts is found in sheep - raising countries, including north america and south america, the entire australia, new zealand, europe, central asia, china, and parts of africa (1, 2, 46). Various parts of body may be involved with hydatid cyst but the liver and lungs are the main locations (7). Skeletal muscle infection is rare, and reported 0.5% 4.7% of all cases of echinococcosis (8). Patients with hydatid cyst are asymptomatic and present at an advanced stage of hydatidosis, when lesions have become extensive (7). Here we report a case of this rare entity of an isolated hydatid cyst of the muscle of the thigh . A 70-years old housewife living in ardoghesh, a village in neyshabur, northeast of iran, was admitted to general surgery clinic in feb 2014 because of a painless mass in the back of her left thigh . She had a history of removing hydatid cyst surgery in her left thigh from three years ago . Laminated layers of hydatid cysts were observed and two round masses was successfully removed (fig . Hydatid cyst with laminated layer (original) gross pathology showed two soft cystic masses (134/54/5 cm and other 1744 cm in diameter) that contained gelatinous material and multiple daughter cysts . In histopathologic examination cross section of a hydatid cyst with laminated layer and germinal layer, brood capsules containing multiple protoscolices, hooklets are considered as diagnostic keys (h&e). Pathological examination confirmed diagnosis as hydatid cyst of thigh (fig . 2, 3). Hydatid cyst in muscle (original) hydatid cyst in muscle showing germinal layer (original) hydatid disease is a very serious problem in sheep - raising countries caused by tapeworms belonging to the class cestoda, in the family taeniidae, of the genus echinococcus . They measure 3 mm to 6 mm long when mature and lives in intestine of carnivores, particularly dogs and other canines, as definitive hosts . Many mammals may serve as intermediate hosts, but herbivorous species are most likely to become infected by eating eggs on contaminated herbal material . Humans are seldom involved as accidental intermediate hosts in these cases and infected by accidentally ingestion of echinococcus spp . Hydatidosis is a serious public health problem in some parts of the world (4, 10). Khorasan province, located in the northeastern part of iran had the highest incidence rate for hydatidosis (11). Although the incidence of hydatidosis has decreased because of education and control programs, there are still concerns in some parts of the world (12). Hydatid cyst is most commonly found in the liver and lung, while they can occur in other organs including muscle, brain, eye, spleen, kidney, orbit, lymphatic glands, myocardium, tonsil, pancreas, skin, ovary, uterus and parotid glands (2, 1317). Hydatidosis are usually asymptomatic until adolescence due to the slow growing process of the parasite in tissues such as muscle and bone, although it can be acquired at any age (18). Incidence rate of musculoskeletal hydatidosis is not clear . Some reports showed an incidence of musculoskeletal echinococcosis including involvement of subcutaneous tissue as 0.5%4.7% among all cases of hydatid disease and soft - tissue hydatid cysts occur in 2.3% of cases reported from endemic areas (8, 19). Muscles provide a poor environment for the parasite because of the presence of lactic acid and mechanical factors, such as contractile activity, may make encystment less likely (1820). It may mimic any soft tissue tumor such as abscess, synovial cyst, and malignant tumor . Before biopsy of cyst, diagnosis of hydatidosis should be confirmed to avoid leakage of cyst contents and the accompanying risks of anaphylaxis (20). Hydatidosis in soft tissues may present with a variety of patterns and recognizing them is necessary in diagnosis (18). In our presented case, preoperative diagnosis was malignant neoplasm and after surgery, hydatidosis was confirmed . Rokni yazdi et al . Noted left thigh hydatidosis in a 50-years - old housewife living in a village in zanjan, northwestern iran (18). Noted left thigh hydatidosis in a 50-year - old woman from rural area around rasht city with no history of trauma, fever, or weight loss (20). In conclusion, the hydatid cyst can present in any part of the body and no part is protected . The infestation may resemble a soft tissue tumor in the muscle and therefore in endemic area of hydatidosis, hydatid cyst should be considered as differential diagnosis of any soft tissue mass . In this case,
Since the 1970s, incidence rate and mortality for cancer have been rising in china . Cancer has become the first killer of human in the 21st century . Based on the data of china national cancer center, the crude morbidity rate of cancers in 2012 has reached 264.85/100,000 and the crude mortality rate of cancers has reached 161.49/100,000 (1). About 13% of death in chinese residents (1 out of 78 deaths) was caused by cancer (2). Cancer not only seriously affects the health of the residents, but also causes heavy economic burden to family and society . The prevention and treatment for cancer has become a major public health problem in china . In 2010, the total expenditure for the prevention and treatment of cancer was 94.84 billion rmb (13.79 billion us dollars), accounting for 5.14% of the total health expenses and 0.24% of gross domestic product (gdp) in china at the same period (3). In 2015, the total expense for cancer in tumor special hospitals reached 50.76 billion rmb (7.38 billion us dollars) (4). Information on hospital expenditure is an important input to the economic and financial analyses of many types of health interventions, and is also essential for budgeting and planning exercises . Previous studies (5,6) mainly focused on the quantification of the cost of cancer, which is an absolute number and cannot account for the cancer - caused proportion of health resources in various diseases . Moreover, analysts estimating the costs of health interventions requiring hospitalization often cut corners because they lack data and the costs of undertaking full step - down costing studies are high . They sometimes use the costs taken from a single hospital; and sometimes use simple rules of thumb for allocating total hospital costs (7,8). To fill the gap, for the first time, we analyzed the proportion of cancer - caused hospitalization expenses in total hospitalization expenses from the national authoritative data, explored the influencing factors of the proportion, and provided effective data information for more rational utilization of health resources, so as to control unreasonable medical expenses, reduce the economic burden, and promote health care system reform and the management of health care costs . Furthermore, this study could be used to understand possible influencing factors of inpatient expenditure pattern for cancer (iepc), and provides evidence for future studies to predict costs in regions where data were not available . The related information on inpatient expenditure for cancer in china in 2015 is from the inpatient medical record home page (imrhp) of general hospitals, whose main contents include patients demographic information, code of diagnosis, code of surgery, results of treatments, hospitalization expenses, etc . The imrhp was quarterly reported by the general and special hospitals in the secondary and above levels through the health statistics and report system of center for health statistics and information (chsi), national health and family planning commission, china . Hospitals update their information as long as the information changed through the health statistics and report system of chsi . Hospital information mainly includes hospital s property, level, number of staff, number of beds, situation of equipment, volume of medical services, etc . Provincial data, including gdp per capita, the proportion of urban population, age structure, sex ratio and so on, are from china health and family planning statistical yearbook 2016 . Every year before march, national chsi checks the completeness and validity of imrhp and hospital information of last year by provincial level and informs the provincial chsis of the results of data quality control . Besides, national chsi carries out data quality supervision and inspection regularly, and helps the provinces with poorer data quality to improve their data s completeness and validity . Considering the possible clustering in iepc, we used multilevel model to explore the influencing factors of ratio . As the ratio after logarithmic transformation obeys normal distribution, we proposed two - level (hospital level and province level) linear regression model . Cary, usa) to clean data, explore analysis and choose explanatory variables, and modeled by mlwin (version 2.02; multilevel models project institute of education, london, uk). To keep the consistency with world health organizaiton relative study (9) we applied linear regression model to estimate the influencing factors of iepc . To better explain the potential clustering and differences of the proportions, we proposed two - level linear regression model, with hospital as the low level and province as the high level, taking account of the potential homogeneity with similar disease distribution in provinces (10). As the outcome variable, iepc s numerator is the total hospitalization expenditure of patients primarily diagnosed as cancer (international classification of diseases 10: c00-c97) in a hospital and denominator is the total hospitalization expenditure in a hospital . A total of 40.76 million imrhp from 2,886 general hospitals were included in the analysis . We took almost all observed relevant variables of hospital and province into consideration in order to analyze influencing factors of inpatient expenditure pattern . One was the possible application of the model in predicting iepc in regions without imrhp for the future, namely the availability of data . Firstly, data included in this paper are available all over the country because of the availability of national hospital information database and china health and family planning statistical yearbook . Adamet al . Analyzed influencing factors of the ratio of the ward - specific cost to the total cost by the explanatory variables: province, number of staff, number of beds and occupancy rate (11), and modeled by gdp per capita, occupancy rate, hospital beds, public or not, etc ., to analyze influencing factors of variation in the cost of inpatient stays versus outpatient visits in hospitals (9). We referenced variables used by adam, but tried more variables to explore possible correlations . In addition to the aforementioned variables, hospital level, age - standardized mortality, floating population, age structure and sex ratio were also brought into model . The multilevel model is as follows: \begin{document} $\begin{aligned} {y_{ij}} = & {\beta _ 0}_{ij} + {\beta _ 1}grad{e_{ij}} + {\beta _ 2}propert{y_{ij}} + {\beta _ 3}be{d_{ij}} + \\ & {\beta _ 4}butilizatio{n_{ij}} + {\beta _ 5}g\!\;\!d\!\;\! {p_j} + {\beta _ 6}s\!m\!ortalit{y_j} + \\ & {\beta _ 7}oprovinc{e_j} + {\beta _ 8}ym\!iddl{e_j} + {\beta _ 9}ol{d_j} + {\beta _ {10}}s\!ex\! {r_j} \end{aligned} $\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\! {\beta _ {0ij}} = {\beta _ 0} + {u_{0j}} + {e_{0ij}}$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \left [{{u_{0j}}} \right] \!\sim\! N\left ({0,{\rm {}} \sigma _ {{u_0}}^2} \right)$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \left [{{u_{0ij}}} \right] \!\sim\! N\left ({0,{\rm {}} \sigma _ {{e_0}}^2} \right)$\end{document} the related information on inpatient expenditure for cancer in china in 2015 is from the inpatient medical record home page (imrhp) of general hospitals, whose main contents include patients demographic information, code of diagnosis, code of surgery, results of treatments, hospitalization expenses, etc . The imrhp was quarterly reported by the general and special hospitals in the secondary and above levels through the health statistics and report system of center for health statistics and information (chsi), national health and family planning commission, china . Hospitals update their information as long as the information changed through the health statistics and report system of chsi . Hospital information mainly includes hospital s property, level, number of staff, number of beds, situation of equipment, volume of medical services, etc . Provincial data, including gdp per capita, the proportion of urban population, age structure, sex ratio and so on, are from china health and family planning statistical yearbook 2016 . Every year before march, national chsi checks the completeness and validity of imrhp and hospital information of last year by provincial level and informs the provincial chsis of the results of data quality control . Besides, national chsi carries out data quality supervision and inspection regularly, and helps the provinces with poorer data quality to improve their data s completeness and validity . Considering the possible clustering in iepc, we used multilevel model to explore the influencing factors of ratio . As the ratio after logarithmic transformation obeys normal distribution, we proposed two - level (hospital level and province level) linear regression model . Cary, usa) to clean data, explore analysis and choose explanatory variables, and modeled by mlwin (version 2.02; multilevel models project institute of education, london, uk). To keep the consistency with world health organizaiton relative study (9) we applied linear regression model to estimate the influencing factors of iepc . To better explain the potential clustering and differences of the proportions, we proposed two - level linear regression model, with hospital as the low level and province as the high level, taking account of the potential homogeneity with similar disease distribution in provinces (10). As the outcome variable, iepc s numerator is the total hospitalization expenditure of patients primarily diagnosed as cancer (international classification of diseases 10: c00-c97) in a hospital and denominator is the total hospitalization expenditure in a hospital . A total of 40.76 million imrhp from 2,886 general hospitals were included in the analysis . We took almost all observed relevant variables of hospital and province into consideration in order to analyze influencing factors of inpatient expenditure pattern . One was the possible application of the model in predicting iepc in regions without imrhp for the future, namely the availability of data . Firstly, data included in this paper are available all over the country because of the availability of national hospital information database and china health and family planning statistical yearbook . Adamet al . Analyzed influencing factors of the ratio of the ward - specific cost to the total cost by the explanatory variables: province, number of staff, number of beds and occupancy rate (11), and modeled by gdp per capita, occupancy rate, hospital beds, public or not, etc ., to analyze influencing factors of variation in the cost of inpatient stays versus outpatient visits in hospitals (9). We referenced variables used by adam, but tried more variables to explore possible correlations . In addition to the aforementioned variables, hospital level, age - standardized mortality, floating population, age structure and sex ratio were also brought into model . The multilevel model is as follows: \begin{document} $\begin{aligned} {y_{ij}} = & {\beta _ 0}_{ij} + {\beta _ 1}grad{e_{ij}} + {\beta _ 2}propert{y_{ij}} + {\beta _ 3}be{d_{ij}} + \\ & {\beta _ 4}butilizatio{n_{ij}} + {\beta _ 5}g\!\;\!d\!\;\! {p_j} + {\beta _ 6}s\!m\!ortalit{y_j} + \\ & {\beta _ 7}oprovinc{e_j} + {\beta _ 8}ym\!iddl{e_j} + {\beta _ 9}ol{d_j} + {\beta _ {10}}s\!ex\! {r_j} \end{aligned} $\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\! {\beta _ {0ij}} = {\beta _ 0} + {u_{0j}} + {e_{0ij}}$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \left [{{u_{0j}}} \right] \!\sim\! N\left ({0,{\rm {}} \sigma _ {{u_0}}^2} \right)$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \left [{{u_{0ij}}} \right] \!\sim\! N\left ({0,{\rm {}} \sigma _ {{e_0}}^2} \right)$\end{document} considering the possible clustering in iepc, we used multilevel model to explore the influencing factors of ratio . As the ratio after logarithmic transformation obeys normal distribution, we proposed two - level (hospital level and province level) linear regression model . Cary, usa) to clean data, explore analysis and choose explanatory variables, and modeled by mlwin (version 2.02; multilevel models project institute of education, london, uk). To keep the consistency with world health organizaiton relative study (9) we applied linear regression model to estimate the influencing factors of iepc . To better explain the potential clustering and differences of the proportions, we proposed two - level linear regression model, with hospital as the low level and province as the high level, taking account of the potential homogeneity with similar disease distribution in provinces (10). As the outcome variable, iepc s numerator is the total hospitalization expenditure of patients primarily diagnosed as cancer (international classification of diseases 10: c00-c97) in a hospital and denominator is the total hospitalization expenditure in a hospital . A total of 40.76 million imrhp from 2,886 general hospitals were included in the analysis . We took almost all observed relevant variables of hospital and province into consideration in order to analyze influencing factors of inpatient expenditure pattern . One was the possible application of the model in predicting iepc in regions without imrhp for the future, namely the availability of data . Firstly, data included in this paper are available all over the country because of the availability of national hospital information database and china health and family planning statistical yearbook . Adamet al . Analyzed influencing factors of the ratio of the ward - specific cost to the total cost by the explanatory variables: province, number of staff, number of beds and occupancy rate (11), and modeled by gdp per capita, occupancy rate, hospital beds, public or not, etc ., to analyze influencing factors of variation in the cost of inpatient stays versus outpatient visits in hospitals (9). We referenced variables used by adam, but tried more variables to explore possible correlations . In addition to the aforementioned variables, hospital level, age - standardized mortality, floating population, age structure and sex ratio were also brought into model . The multilevel model is as follows: \begin{document} $\begin{aligned} {y_{ij}} = & {\beta _ 0}_{ij} + {\beta _ 1}grad{e_{ij}} + {\beta _ 2}propert{y_{ij}} + {\beta _ 3}be{d_{ij}} + \\ & {\beta _ 4}butilizatio{n_{ij}} + {\beta _ 5}g\!\;\!d\!\;\! {p_j} + {\beta _ 6}s\!m\!ortalit{y_j} + \\ & {\beta _ 7}oprovinc{e_j} + {\beta _ 8}ym\!iddl{e_j} + {\beta _ 9}ol{d_j} + {\beta _ {10}}s\!ex\! {r_j} \end{aligned} $\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\! {\beta _ {0ij}} = {\beta _ 0} + {u_{0j}} + {e_{0ij}}$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \left [{{u_{0j}}} \right] \!\sim\! N\left ({0,{\rm {}} \sigma _ {{u_0}}^2} \right)$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \left [{{u_{0ij}}} \right] \!\sim\! N\left ({0,{\rm {}} \sigma _ {{e_0}}^2} \right)$\end{document} i: the first / low level, hospital level;j: the second / high level, province level;yij: nature logarithmic of iepc in a hospital;k: fixed coefficient of each explanatory variable, k=1, 2, 3 10;gradeij: hospital level;propertyij: hospital property;bedij: number of beds in a hospital;butilizationij: bed utilization in a hospital;gdpj: gdp per capital in a provi - nce;smortalityj: age - standardized mortality for cancer in a province;oprovincej: proportion for inpatient outside of home province;ymiddlej: ratio of population aged 1564 years old in a province;oldj: ratio of population 65 years old in a province;sexrj: sex ratio in a province; \begin{document}$\sigma _ {{u_0}}^2$\end{document}: between province variance; \begin{document}$\sigma _ {{e_0}}^2$\end{document}: within province variance . We analyzed 40.76 million imrhp of 2,886 hospitals in 30 provinces of china in 2015 except tibet because of few data on tibet . There was a big difference in the number of the eligible reported imrhp among provinces in china in 2015 . The iepc of the total inpatient records was 11.46% in china in 2015, ranging from 3.03% to 19.61% across provinces . Top three provinces on iepc was fujian, jiangsu and zhejiang, whose iepc were 19.61%, 18.29% and 16.35%, respectively . Guizhou, shandong and shaanxi had the least proportion of iepc, which were 3.03%, 3.92% and 4.74%, respectively . Due to the skewed distribution for iepc, we used median and inter - quartile range as presented intable 1 . The distribution for inpatient medical record home page (imrhp) in china, 2015 . Iepc of 30 provinces in china, 2015 the number of public hospitals was approximately 6 times as many as that of private hospitals, while the median of iepc% of public hospitals was about 2 times as high as that of private hospitals . The number of secondary hospitals amounted to 2,071, which was far more than the numbers of primary and tertiary hospitals . For iepc%, tertiary hospitals were the highest, secondary hospitals were about a quarter of tertiary hospitals, and the remaining was approximately one seventh of the tertiary . Despite middle regions had the least number of hospitals than the west and the east regions, the median of iepc% of east regions was slightly higher than that of the middle, and that of west regions was the least (table 2). Iepc in different classification in china, 2015 single factor analysis and correlation analysis were carried out for explanatory variables . We established a variance covariance matrix to analyze the correlations in all variables, which were divided into four categories: property, level, scale and efficiency of hospital . The results of correlations showed that staff and income variables are highly correlated with beds (pearson correlation coefficient> 0.9). The highly correlated variable (pearson correlation coefficient> 0.75) with the rest variables in each category was selected as representative . Finally, we selected four representative types: hospital property (1 is a public hospital, 0 is a private hospital), hospital level (1, 2, 3 are primary, secondary and tertiary hospital, respectively), number of beds in a hospital, and bed utilization in a hospital (numerator is bed - days used by patients and denominator is total bed - days in hospital). Variance covariance matrix for provincial variables showed that the region (the east, middle and west of china) and life expectancy per capita are highly correlated with gdp per capita . According to single factor liner regression model, the population variable had no statistical significance . In the end, the provincial variables include: gdp per capital, age - standardized mortality for cancer (12), proportion for inpatient outside of home province (numerator is the total number of trans - provincial inpatients and denominator is the total number of provincial inpatients), ratio of population aged 1564 years old, ratio of population 65 years old, and sex ratio . As shown in model 0 without any explanatory variables, also called zero model, second - level s residual had statistically significant difference, suggesting the iepc in a province having homogeneity . When we put the variables successively in the model, we found that hospital level and number of beds in a hospital had statistical significance, and the rest of the explanatory variables showed no significance . The residual normal figures of hospital level and province level were similar to a straight line, indicating that the hypothesis of first level and second level s residual obeying normal distribution is reasonable and the application of the above model is correct . Fixed coefficient of each variable is presented intable 3 . The results of final model are as follows: \begin{document} $\begin{aligned} \quad {y_{ij}} = & {\beta _ 0}_{ij} + 0.475grad{e_{ij}} {\rm {\,-\,}} 0.036propert{y_{ij}} \,+ \\ & 0.001be{d_{ij}} + 0.032butilizatio{n_{ij}} {\rm {\,-\,}} {\rm{0}}.{\rm{000}}g\!d\! {p_j} \,+ \\ & 569.245s\!m\!ortalit{y_j} + 1.520oprovinc{e_j} \,+ \\ & 0.056ym\!iddl{e_j} + 0.117ol{d_j} + 0.011sex{r_j} \end{aligned} $\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \quad {\beta _ {0ij}} = {\rm {-}} 12.390 + {u_{0j}} + {e_{0ij}}$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \quad \left [{{u_{0j}}} \right] \!\sim\! N\left ({0,{\rm {0}}{\rm{.153}}} \right)$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!! N\left ({0,{\rm {0}}{\rm{.986}}} \right)$\end{document} two - level linear regression model on influencing factors of iepc we analyzed 40.76 million imrhp of 2,886 hospitals in 30 provinces of china in 2015 except tibet because of few data on tibet . There was a big difference in the number of the eligible reported imrhp among provinces in china in 2015 . The iepc of the total inpatient records was 11.46% in china in 2015, ranging from 3.03% to 19.61% across provinces . Top three provinces on iepc was fujian, jiangsu and zhejiang, whose iepc were 19.61%, 18.29% and 16.35%, respectively . Guizhou, shandong and shaanxi had the least proportion of iepc, which were 3.03%, 3.92% and 4.74%, respectively . Due to the skewed distribution for iepc the distribution for inpatient medical record home page (imrhp) in china, 2015 . Iepc of 30 provinces in china, 2015 the number of public hospitals was approximately 6 times as many as that of private hospitals, while the median of iepc% of public hospitals was about 2 times as high as that of private hospitals . The number of secondary hospitals amounted to 2,071, which was far more than the numbers of primary and tertiary hospitals . For iepc%, tertiary hospitals were the highest, secondary hospitals were about a quarter of tertiary hospitals, and the remaining was approximately one seventh of the tertiary . Despite middle regions had the least number of hospitals than the west and the east regions, the median of iepc% of east regions was slightly higher than that of the middle, and that of west regions was the least (table 2). We established a variance covariance matrix to analyze the correlations in all variables, which were divided into four categories: property, level, scale and efficiency of hospital . The results of correlations showed that staff and income variables are highly correlated with beds (pearson correlation coefficient> 0.9). The highly correlated variable (pearson correlation coefficient> 0.75) with the rest variables in each category was selected as representative . Finally, we selected four representative types: hospital property (1 is a public hospital, 0 is a private hospital), hospital level (1, 2, 3 are primary, secondary and tertiary hospital, respectively), number of beds in a hospital, and bed utilization in a hospital (numerator is bed - days used by patients and denominator is total bed - days in hospital). Variance covariance matrix for provincial variables showed that the region (the east, middle and west of china) and life expectancy per capita are highly correlated with gdp per capita . According to single factor liner regression model, the provincial variables include: gdp per capital, age - standardized mortality for cancer (12), proportion for inpatient outside of home province (numerator is the total number of trans - provincial inpatients and denominator is the total number of provincial inpatients), ratio of population aged 1564 years old, ratio of population 65 years old, and sex ratio . As shown in model 0 without any explanatory variables, also called zero model, second - level s residual had statistically significant difference, suggesting the iepc in a province having homogeneity . When we put the variables successively in the model, we found that hospital level and number of beds in a hospital had statistical significance, and the rest of the explanatory variables showed no significance . The residual normal figures of hospital level and province level were similar to a straight line, indicating that the hypothesis of first level and second level s residual obeying normal distribution is reasonable and the application of the above model is correct . The results of final model are as follows: \begin{document} $\begin{aligned} \quad {y_{ij}} = & {\beta _ 0}_{ij} + 0.475grad{e_{ij}} {\rm {\,-\,}} 0.036propert{y_{ij}} \,+ \\ & 0.001be{d_{ij}} + 0.032butilizatio{n_{ij}} {\rm {\,-\,}} {\rm{0}}.{\rm{000}}g\!d\! {p_j} \,+ \\ & 569.245s\!m\!ortalit{y_j} + 1.520oprovinc{e_j} \,+ \\ & 0.056ym\!iddl{e_j} + 0.117ol{d_j} + 0.011sex{r_j} \end{aligned} $\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \quad {\beta _ {0ij}} = {\rm {-}} 12.390 + {u_{0j}} + {e_{0ij}}$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!! N\left ({0,{\rm {0}}{\rm{.153}}} \right)$\end{document} \begin{document} $\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!! N\left ({0,{\rm {0}}{\rm{.986}}} \right)$\end{document} two - level linear regression model on influencing factors of iepc studying influencing factors of iepc in china can provide evidence for more reasonable allocation of limited health resources . The results show that cancer takes up a large amount of health resources, strengthening the importance of greater attention . Additionally, iepc in a high level has clustering, namely that there is heterogeneity between provinces and similarity within a province . Model results demonstrate that hospital level and number of beds are the influencing factors of iepc . We can see that the higher the level of hospitals, the higher the value of iepc . This result can be explained by the fact that the patients with severe diseases tend to visit the hospitals with higher level . Although the levels can reflect the ability of diagnosis and treatment of hospitals in a certain level, there still is a high variation of inpatient volume among hospitals in the same level . Our study shows that the expenditure of cancer was concentrated in hospitals with more beds . Briefly, rising one level of the hospital led to the increase of 0.475 natural logarithm units of iepc averagely . The number of beds increasing 1,000 each made the natural logarithm of iepc increase one unit averagely . Although the rest of the explanatory variables cannot influence the iepc, they can explain the differences between provinces to some extent, including gdp per capita, age - standardized mortality for cancer, proportion for inpatient outside of home province and ratio of population aged 1564 years old . The final model can explain 50.65% of the variation, and the independent variables explained the model well (13). To our knowledge, this is the first study exploring the iepc using national authoritative data to acquire the proportion of cancer in various diseases in china . Firstly, some influencing factors (such as the price of cancer treatment) on iepc could not be included in the model due to the limitation of data accessibility . Secondly, the variation of data completeness among different levels of hospitals and different regions may influence our final model . However, this is still the inpatient expenditure pattern study with the largest data sample . Our study showed that a considerable proportion of iepc variation could be explained by the differences of hospital level and number of beds . It implied that it is possible to estimate disease - specific ratio of inpatient expense taking account of key influencing factors in china . Furthermore, this study is an input to economic and financial analyses and provides evidence for future study on the national economic burden of cancer . What s more, the influencing factors obtained from this study can predict the ratio in the area where data are not available.
West nile encephalitis is a mosquito - borne disease endemic to africa, asia, australia, and europe and the west nile virus is a single - stranded rna virus which belongs to the japanese encephalitis serogroup of flaviviruses (1). It could be accompanied with various neurologic symptoms and signs including headache, mental confusion, optic neuritis, and muscle weakness (2, 3, 4). After the first two cases of west nile encephalitis were diagnosed in 1999 in new york (5), many neurological abnormalities were noted in patients of west nile encephalitis, and the flaccid muscle weakness is a distinctive feature of this disease (2, 3, 6). Until now, west nile encephalitis was never diagnosed in korea, and it is the first report of west nile encephalitis in korea . A 58-yr - old man was admitted with headache for 2 months in june 2012 . The patient had been on a business trip in guinea, west africa since 7 months ago . He denied other symptoms such as nausea, vomiting, neck stiffness, and dizziness . The headache and cognitive impairment had been worsening and accompanied general weakness and weight loss 10 kg for 2 months . He had no hypertension, cardiac diseases, chronic renal disease, and liver disease . He had a 20 pack - year history of smoking and no recent alcohol intake . The patient denied using any medications except for antimalarial drug (sulfadoxine 500 mg, pyrimethamine 25 mg, artemether 80mg / ml) and analgesics in africa . At admission to our hospital, he was afebrile (36.4) and his vital signs were blood pressure 121/78 mmhg, heart rate 97/min, and respiratory rate 20/min with normal findings of physical examinations . Findings on initial neurologic examinations were normal except minimental state examination (mmse) with score 20 . Admission complete blood count revealed hemoglobin of 12.7 g / dl, leukocyte count of 16.2 10/l with 79% neutrophils, and a platelet count of 388 10/l . The patient's calcium level, chemistries, and liver function tests were normal and crp was slightly increased . Analysis of cerebrospinal fluid (csf) demonstrated 0 rbc/l, 214 wbcs/l (with 80% lymphocytes and 12% neutrophils), a protein level of 161.9 mg / dl, a glucose level of 93 mg / dl and ada level of 4.8 . A brain magnetic resonance imaging (mri) revealed multifocal patchy t2 high signal lesions in both temporal lobes, basal ganglia, internal capsule posterior limb, and corona radiata, which suggested encephalitis . Electroencephalogram showed frequent semirrhythmic medium amplitude delta slowing on both hemispheres, with frontal dominancy . On hospital day 1, his body temperature was 38.0 and malaria test was negative . Follow up csf tapping was done on hospital day 3 and 6 rbc/l, 340 wbcs/l (with 67% neutrophils and 28% lymphocytes), a protein level of 164.6 mg / dl, a glucose level of 70 mg / dl and ada level of 6.0 . The patient complained of both leg weakness and the motor strength of bilateral lower extremity was 4+/4 + . He also complained of both leg pain and paresthesia, deep tendon reflex on both lower extremities were areflexia . There was no remarkable finding on nerve conduction study (ncs) and needle electromyogram (emg), but arachnoiditis and myelitis were observed on lumbar spine mri (fig . Polymerase chain reactions (pcr) for hsv, vzv, ebv, cmv, enterovirus, tuberculosis, and mycoplasma pneumonia were negative in csf . Serological test for brucellosis, lyme disease, leptospirosis, hantann virus and tsutsugamuchi disease were negative at admission an additional serological study for specific antibody for west nile virus was done, the total antibody titer of west nile virus was 1:337, and follow up titer after 2 weeks was 1:1,632 in serum by plaque reduction neutralization test . Treatment with interferon- 3mu was started and maintained for 2 weeks . Follow up csf tapping was done after one month later and 9 rbc/l, 9 wbcs/l (with 16% neutrophils and 67% lymphocytes), a protein level of 99.9 mg / dl and a glucose level of 66 mg / dl . Encephalitis with severe muscle weakness is a common symptom and may provide a diagnostic clue . To confirm a diagnosis, it needs identification of west nile virus in serum or csf . Igm antibody is usually detectable only during a few days of illness (7). Another method is the identification of a change by a factor of four or more in the antibody titer or virus - neutralization tests (7), the case was positive for the virus - neutralization tests . Serological test and pcr for other infectious agents were all negative and autoimmune encephalitis such as nmda receptor encephalitis was excluded by clinical symptoms (8). Many studies indicated that the weakness was probably caused by infection and inflammation in the anterior horn cells of the spinal cord and brainstem like poliomyelitis (6, 9, 10). Electrophysiologic studies showed reduced compound muscle action potentials, and normal sensory nerve action potentials, which is distinguished from guillain - barre syndrome (3, 6, 9, 10). A few autopsy studies demonstrated that the inflammatory changes with severe leukocytic inflammation of the anterior horns with striking loss of motor neurons (4, 11). But this case did not present any abnormalities in the electrophysiologic study . He complained about severe sensory symptoms with leg pain and arachnoiditis was observed in lumbar mri . Only a few reports noted a caudal equine enhancement (4) and weakness due to acute radiculitis (12). It's hard to say that his weakness was caused by only radiculopathy, because paralysis of the west nile encephalitis generally results from anterior horn cell damage . But in this case, radiculopathy also contributes the motor weakness of the west nile encephalitis . At present there is no established treatment for west nile encephalitis and basic treatment is a supportive care . But because it's a fatal disease, sometimes it was treated with plasmapheresis or administration of iv immunoglobulin (5). Interferon- has been reported to inhibit west nile virus replication, and some successful cases were reported using of interferon- to treat west nile encephalitis (13). Korea is not an endemic area of west nile virus, but an increasing overseas trip poses a risk of the influx of overseas diseases and may contribute to the global spread of infectious diseases.
Migraine is a common neurological illness worldwide leading to significant effects on morbidity, productivity and quality of life . The anticonvulsant topiramate is known in the western setting to be effective in migraine prevention [24]. These previous studies, of which asians form the minority, have employed dosages at or above 100 mg / day for prophylaxis . However, there is evidence that lower dosages of topiramate may be effective in migraine prevention, particularly in the asian population [57]. To our knowledge, three previous studies comprising koreans, chinese and indians, have addressed the use of topiramate in the asian patients [57]. Two studies had employed dosages of at or above 50 mg / day [5, 6], while the third, utilizing a variable titration method, had suggested that daily dosages as low as 25 mg may be effective . In addition, two other studies involving iranian patients have utilized topiramate dosages up to 50 mg / day and concluded it to be as effective as propanolol and sodium valproate in migraine prophylaxis [8, 9], providing further evidence of its efficacy in asians . In this study, we sought to further clarify dosage issues in a prospective dosing study of migraine patients in the multiracial asian setting . With local ethical committee (singapore general hospital institutional review board) approval, we prospectively screened 82 patients having migraine with or without aura, as defined by the international classification of headache disorders 2nd edition . In particular, we had ensured that patients included should have at least four headache episodes in the preceding 4 weeks, with each episode at least four of ten in severity on the visual analogue scale (vas). 42 were excluded: 7 patients became un - contactable prior to randomization, 23 were screen failures and 12 had withdrawn consent during the study . A total of 40 patients (age range 1864, mean age 38, 30 females, 10 males) were randomized into the study . Patients were recruited in a consecutive fashion from a headache specialist clinic in a tertiary general hospital . Each was briefed upon randomization on how to record symptoms and side effects into the headache diary . Each patient had kept a headache diary 4 weeks prior to randomization, documenting baseline migraine attacks over this period . Patients documented frequency as the number of days with headache in the preceding 4 weeks, duration of the most severe attack in a day in hours, and severity measured as the worst period during an attack on a vas from 0 to 10 cm . During the entire treatment period, they were allowed symptomatic medications such as paracetamol, but triptans and opioids were disallowed . However, any prior use of prophylactic medication of any class at any time documented was an exclusion criterion . This was defined as usage of triptans> 3 days a week, simple analgesics> 4 days a week or narcotic analgesics> 2 days a week . Each patient was randomized to receive 25, 50, 75 or 100 mg of topiramate as an evening dose, with 10 patients in each arm . For the 50 mg arm, patients started on 25 mg for 1 week before continuing with 50 mg for the rest of the study . Similarly, for the 75 mg arm, patients started with 25 mg for 1 week, 50 mg for the second week, and 75 mg for the subsequent weeks . The end - point of the study was 12 weeks after achieving the intended topiramate dosage for that particular treatment arm, and patients returned for follow - up every 4 weeks after starting treatment . Comparisons were made between migraine attacks at baseline and the 4-week period prior to the end - point (fig . 2). It should be noted that upward titration of dosages were gradual and necessary to minimize side effects and drop outs . For the 50, 75 and 100 mg arms, each patient would have received a total of 12 weeks of the intended dose of that particular arm before study end - point was achieved . Statistical calculations were performed with spss for windows package, employing the wilcoxon signed rank, kruskal wallis and pearson s correlation tests . A p value <0.05 was regarded as statistically significant . Of the 40 patients who had completed the trial, no significant differences in baseline headache frequency, duration and severity was found between the four treatment arms . Overall, we found that topiramate significantly reduced headache frequency (headache days) comparing baseline to end - point (wilcoxon signed rank test, z = 3.47, p = 0.001). However, no significant differences were found for headache duration (wilcoxon signed rank test, z = 0.23, p = 0.82) or severity (wilcoxon signed rank test, z = 0.31, p = 0.76). In terms of side effects, paresthesia was by far the most commonly encountered in 55% of patients . However, its incidence was highly correlated with increasing dosage (pearson s correlation, r = 0.96, p <0.001). 1overall headache parameters comparing baseline (time point 1) to study end - point (time point 2)fig . 2flow diagram showing protocol from screening to end of studytable 1summary of most common side effects in 40 patientsside effectdose (number of patients)25 mg50 mg75 mg100 mgparesthesia4567fatigue1132giddiness1211diarrhoea3001nausea1102drowsiness1120table 2summary of results of all recruited patientstimebaselinetime 1time 2time 3frequency (days) 25 mg10.2 (5.1)7.6 (6.4)3.5 (2.2)4.1 (1.9) 50 mg6.9 (2.6)8.1 (4.2)6.5 (4.8)6.8 (5.7) 75 mg8.8 (4.4)6.2 (5.9)5.8 (6.6)3.3 (4.4) 100 mg8.0 (2.5)8.8 (8.2)4.3 (2.4)2.3 (1.5)p = 0.001duration (h) 25 mg8.4 (3.8)9.2 (5.5)10.1 (8.0)6.9 (7.7) 50 mg8.8 (8.6)6.0 (6.1)5.3 (5.3)5.9 (5.4) 75 mg5.3 (4.7)7.7 (6.3)10.9 (2.6)12.3 (8.0) 100 mg7.9 (7.1)6.4 (8.8)7.3 (3.1)8.0 (5.7)p = 0.82severity (vas) 25 mg5.1 (1.2)5.1 (1.9)5.6 (1.7)4.9 (0.8) 50 mg6.3 (1.3)6.0 (0.5)5.1 (1.3)5.8 (1.4) 75 mg5.0 (1.5)4.4 (1.4)4.0 (1.4)4.3 (1.4) 100 mg5.7 (1.2)5.8 (1.5)4.8 (1.4)4.8 (1.4)p = 0.75each row denotes mean and (sd)each time point denotes headache diary data of the preceding 4-week period statistical significance (p <0.05) overall headache parameters comparing baseline (time point 1) to study end - point (time point 2) flow diagram showing protocol from screening to end of study summary of most common side effects in 40 patients summary of results of all recruited patients each row denotes mean and (sd) each time point denotes headache diary data of the preceding 4-week period * statistical significance (p <0.05) to our knowledge, this is the first fixed dose ranging study designed specifically to address this issue employing a simple and straightforward clinical approach . Here, we show that a low daily dose of 25 mg of topiramate was equally effective as 50, 75 and 100 mg in reducing headache frequency . The first study assessed topiramate against placebo had found responder rates (50% reduction in headache frequency) at 39% (50 mg / day), 49% (100 mg / day) and 47% (200 mg / day). However, it is comparable to our study only for headache days, which were significantly reduced for the 100 and 200 mg / day groups only . Another study, employing a similar placebo - controlled protocol, reported fairly similar findings . Our findings can be compared to a previous study comprising only chinese patients, which found that some patients appeared to benefit with doses as low as 25 mg / day, despite the mean dose being 80 mg for that entire study which had employed a physician - led upward dose titration depending on clinical response . As the end results of this and our study were contributed by chinese patients, it should be noted that this study was based on an escalating dose titration determined by the treating physician depending on clinical response, and concomitant prophylactic medications were allowed . A second asian study comprising indian patients had employed topiramate at 50 mg / day in a 4-week crossover protocol . In this study, topiramate was administered at a fixed dose of 50 mg / day for only 4 weeks in a crossover design with lamotrigine . These vital differences likely contributed to incomparable findings with our study . To this end, we are of the opinion that a fixed daily dosing regimen would offer maximal practicality and convenience both for the patient and the treating physician . In terms of adverse events, its incidence was increased with higher dosages, as previously noted in other studies [11, 12]. Hence, lower dosages may potentially benefit patients in terms of better compliance as a result of lower side effect occurrences . In addition as asians display clinically different pharmacology compared to western patients, its is likely that our findings of lower dosage requirements can be explained by these physiological factors [13, 14]. In conclusion, our novel findings highlighting efficacy of a fixed low dose of topiramate of 25 mg / day in migraine prophylaxis also attest to advantages in terms of reduced cost and favourable side effect profile.
Snoring is not a benign symptom . It is associated with an increased risk of hypertension, cardiac arrhythmias, and mortality . Snoring is defined as a sound produced when an individual breaths during sleep due to the turbulence of air passing through the partially obstructed airway . It is considered as one of the most common clinical symptoms of obstructive sleep apnea (osa). Snoring may result in excessive daytime sleepiness, loss of concentration, and psychological disturbances that may eventually cause deterioration of the patient life quality unless treated . This wide range is due to the differences in the populations studied, study design, investigations performed, ethnic group, age, and sex of the subjects . Snoring is potentially related to the increase in the body mass index (bmi), cigarette consumption, ethnic differences, infections, and hypertension . Nocturnal polysomnography is considered the best tool to investigate whether snoring is a health risk . However, it is not widely available and expensive to be used as a screening tool, as it is mostly available only in specialized sleep clinics . Therefore, the use of questionnaires like berlin questionnaire become a simple and effective method of screening for snoring and risk of osa . In addition, many craniofacial features and malocclusions were reported to be associated with snoring . For example; narrow airway at the level of the soft palate and oropharynx, more inferiorly positioned hyoid bone, more protruding maxilla, anterior - posterior discrepancy of maxilla and mandible, class ii malocclusion, increase in over - jet, reduced overbite, narrower upper, and shorter lower dental arch and crowding in the mandibular arch . According to literature, snoring was associated with osa, and the characteristics were complicated with other dentofacial risk features such as facial profile, malocclusion classification, and dental arch morphology . For instance, stellzig - eisenhauer and meyer - marcotty (2010) reported a significant difference between patients with retrognathism and pragmatism in respect to the transverse dimension of the nasopharynx . However, de freitas et al ., found no correlation between obstructions of the upper airway and the frequency of malocclusions . This reflects the discrepancy between the influence of the facial profile, malocclusion classification, and dental arch morphology on airway measurements . Therefore, the aim of this study was to test the null hypothesis that there is no morphological difference in facial profile, malocclusion class, and dental arch morphology in adult yemenis male with and without snoring . Rejection of the null hypothesis might improve our understanding of the influence of snoring on the surrounding dentofacial structures . After the study was approved at the institutional level and informed consent was obtained, a cross - sectional study was carried out among male students and employees at the university of science and technology . For the prevalence study, the sample size was calculated using power and sample size software (5.2 mb version 2.1.31 .) Which indicated 840 subjects is required for this study . When the participants agreed to participate in the study, a consent form was given along with a set of the sleep behavior questionnaire (berlin questionnaire). The inclusion criteria were: age 18 and above, healthy individuals without any diseases or congenital abnormalities and the presence of at least six teeth in each maxillary and mandibular arch . Each selected subject undergone clinical examination, upper and lower arch impression, and bmi calculation . Totally, 150 participants (75 snorers: 9 employees, 66 students) and (75 nonsnorers: 8 employees, 67 students) were able to complete their required records . Bmi was calculated from the patient's height and weight in standard units (kg / m), and neck circumference (nc) was measured at the level of the thyroid cartilage . The facial profile was assessed by classifying the facial profile into convex, concave, and straight . Assessment of arch morphology and palatal morphology were carried out as following: arches were classified as constricted and nonconstricted . The dental arch constriction was defined as the presence of two or more maxillary posterior teeth in edge to edge cuspal relationship with their antagonists, or in a frank cross bite . The palatal morphology divided into u - shaped (normal) and v - shaped (constricted). Maxillary constriction was defined by the presence of all the following features: a narrow and high palatal vault; corresponding narrow arch form and unilateral or bilateral buccal tilting of the maxillary alveolar arches with posterior teeth in crossbite or edge to edge relationship with the lower teeth . Assessment of dental cast parameters were completed by taken dental impression and fabricating a set of upper and lower models . Alginate impression material (zhermack, germany) mixed according to the manufacturer's instructions . The following measurements were recorded using digital calipers: inter - canine distance; inter - premolar distance; intermolar distance; arch length; palatal cast depth . Out of 840 questionnaires distributed, 700 were returned (77 employees and 623 students); the response rate was 80% . Nine were employees (11.7%), and 105 were students (16.9%). Totally, 150 participants (75 snorers) and 75 nonsnorers were able to complete their required records and, therefore, were included in the comparison study . Table 1 shows the demographic profile of 150 subjects who were males with mean age of 24 4 years, mean height of 167 6 cm, mean weight of 63 10 kg, mean bmi of 22.5 3 kg / m, and mean nc 36 2.6 cm . Clinical observation of snorers and non - snorers extra - oral variables accordingly, the mean neck size was found to be significantly greater for the snorer group (36.6 2.5) than nonsnorer group [35.4 2.6; p <0.007; table 1]. Clinical examination shows that the most frequent finding among snorer groups when compared with the nonsnorer group were straight profiles (52.0%), class i malocclusion (74.7%), and v palatal shape (16.0%), respectively [table 2]. The chi - square test revealed a significant difference among the v - shape palatal morphology (p <0.034), but no significance was found in terms of the facial profiles; malocclusion class or arch morphology . In addition, the upper arch length was significantly shorter (35.6 2.5) in the snorer subjects than the nonsnorer group (36.4 2.5; p <0.038), and the inter- first upper premolar distance was significantly narrower (39.6 2.6) in the snorer subjects than in the nonsnoring group [40.8 2.9; p <0.013; table 3]. Orthodontic observation of facial profile, malocclusion class, arch morphology and palatal shape orthodontic observation of dental cast measurements (meansd) in snorers and non - snorers the present study was carried out to determine the prevalence of snoring among male adult at university population setting and to compare the craniofacial features of an adult with and without snoring using clinical examination . In addition, the clinical examination showed that the snoring in our adult university population manifested a significantly different craniofacial feature, such as a v - shaped palate; higher nc; lower upper arch length; and a decrease in the inter- first upper premolar distance . Based on our prevalence of snoring result, it seems that the prevalence of snoring in our sample is slightly higher from the average of snoring prevalence quoted earlier but lesser compared with the other studies in student population . Table 4 summarized previous studies of snoring among the student population . Comparing our result to malaysian medical students, our result showed a higher percentage of snoring, which might be due to a mixture of students and employees in our sample; different age; and bmi and diverse definition of snoring . Concluded that snoring was prevalent in student population, and male gender showed a trend as an independent predictor for snoring, which is similar to our result . Summery of snoring among the student population (from most to least severe) when we compared our result with the general adult population findings, our result is slightly lower than the prevalence of snoring reported by middle - eastern studies (52.3% saudi and 28.7% jordanian bahammam et al . And khassawneh et al ., however, in asian adults, it was found that the prevalence of snoring is higher than the western population study . Nevertheless, it should be understood that the prevalence of snoring does not necessarily indicate the development of obstructive complications but these findings do highlight the need for awareness about possible complications . The results of this current study, indicate the nc is significantly greater for the snoring group than for the nonsnorer group . This finding supports the view that nc is one of the significant risk factors for yemeni snorer, which is in good agreement with findings obtained in the chinese and japanese populations . Increased nc even in the absence of obesity in our subjects, may draw our attention on the importance of the fat tissue around the neck in nonobese individuals . Furthermore, snoring and greater nc were also considered as useful clinical predictors of osa . Clinical examination findings indicate that the most frequent finding among snorer groups were straight profiles (52.0%), class i malocclusion (74.7%), and v palatal shape (16.0%), respectively . Even though, there is no relationship between upper airway obstruction and the type of malocclusion were found but it has been reported that the upper pharyngeal width in the subjects with class i and class ii malocclusions with the vertical growth patterns were significantly narrower than in the normal growth pattern group . Therefore, this makes the orthodontist more alert to enquire about snoring even in class i malocclusion and straight profile patients especially with vertical growth patterns . On the other hand, it has been reported that convex profiles (71.7%), class ii malocclusion (51.7%), and v palatal shape (53.3%) were frequent features in an asian osa sample . This in contrast to our current study, and this could be related to the difference between the osa as a general term while the snoring is one of the osa symptoms and to the differences in the cranial base morphology between asians and caucasian . Using dental examination, we found statistically significant difference between yemeni snorers and nonsnorer in a v - shape palatal arch; the arch length and the inter- first premolar distance . In view of the fact that the roof of the mouth is also the floor of the nose, a narrow arch can infringe on the nasal cavity space therefore, maxillary constriction, in addition to his responsibility as one of the important factors for determining nasal airway properties, it can also contribute to lateral narrowing of the upper oral cavity and a low tongue posture which consequently narrow the retroglossal region . This may provide a reasonable explanation for the narrowing of the posterior airway space seen in osa patients . The v palatal shape was also included as one predictive morphometric model for osa, and the model illustrates the potential value of physical and dental examination . For instance, there is a possibility of under diagnosis of snoring when a standard full night polysomnography test is not administered . However, berlin questionnaire can still be considered as an effective and inexpensive way for screening snoring . Another limitation of this study is the fact that females were not included . For cultural factors, this study suggests that snoring among our university population is associated with increased nc; v - shape palatal morphology; the short upper arch length; and narrow inter- first upper premolar distance . Clinically, increased nc even in the absence of obesity may draw our attention to the importance of the fat tissue around the neck in nonobese individuals . In addition, the orthodontist and other health care should be more alert to enquire about snoring, take the complaint of snoring seriously, and perform a thorough clinical and orthodontic evaluation even in class i malocclusion and straight profile patient especially college - aged male adult with vertical growth patterns . Furthermore, on the basis of clinical and orthodontic observation that have been recognized in this study, we believe that those observation features could be used to improve our understanding of the influence of snoring on the surrounding dentofacial structures of the adult university population and may provide valuable screening information in the identification of patients with undiagnosed osa . Further studies are needed to find other clinical and orthodontic features in a diverse university population with different ethnic and sex trends in regards to snoring patterns . Is associated with v - shape palatal morphology, increased nc, and decrease in the upper arch length and inter- first upper premolar distance.
It is the most common bacterial cause of foodborne disease in the usa, causing an estimated 1 million illnesses and 400 deaths annually . In the past, the path from farm to table was shorter and food distribution was more localized . As a result, when foodborne disease outbreaks occurred they were typically local outbreaks in which large numbers of people became ill in one jurisdiction . These outbreaks were normally identified by the affected group with a local investigation, usually resulting from a local food - handling error, and leading to a local solution . While localized outbreaks continue to occur in the usa,, there are a small number of cases in many jurisdictions, detected by laboratory - based subtype surveillance, leading to a multistate or country investigation, and these are usually the result of an industrial contamination event . Driving this change are several factors including a transformation in global food production that has resulted in food being distributed over large distances as well as increased integration and consolidation of agriculture and food production . Processed foods, which contain multiple ingredients, are recognized increasingly as vehicles in salmonella outbreaks [24, 5]. It can be difficult to identify the source of the outbreak when the contaminant is in a wide range of foods, particularly when cases are geographically dispersed and the contamination may be at a low level or not evenly dispersed in product . This study describes an outbreak investigation that identified an ingredient as the source of contamination in multiple food items . State public health laboratories are an integral part of routine surveillance for the identification of foodborne disease outbreaks . These laboratories receive isolates from clinical laboratories for salmonella serotyping and subtyping by pulsed - field gel electrophoresis (pfge). State laboratories submit pfge patterns to pulsenet, the national molecular subtyping network for foodborne disease surveillance . For this investigation, a case was defined as a person with a culture - confirmed salmonella montevideo infection in the usa with illness onset between 1 july 2009 and 1 april 2010 and xbai restriction enzyme pfge pattern jixx01.0011 reported to pulsenet . A probable case was defined as a person with a culture - confirmed salmonella infection in the usa with illness onset between 1 july 2009 and 1 april 2010 and serotype and pfge results pending . To identify possible sources of infections, cdc worked with public health officials in many states to develop a structured hypothesis - generating questionnaire for case - patient interviews . This questionnaire asked whether patients had contact with more than 300 foods and other exposures in the week before illness onset . We also conducted intensive open - ended interviews over the phone that enquired about all foods eaten, travel, grocery stores, and restaurants visited in the week preceding illness . At warehouse stores, members must use their membership cards to purchase products; this creates a record of customer purchases . We used membership records in this investigation to try and identify a common food purchased by ill persons . Ill persons who reported shopping at a warehouse store gave consent to obtain purchase records based on their membership number . Initially, the washington state department of health (wadoh), obtained information from customer purchase records at multiple locations of a national warehouse chain . Subsequently, as the investigation continued, state health departments and cdc collected membership cards from additional store chains . To identify associations between exposures and illness, we conducted a multistate case - control study . Controls were well persons interviewed by telephone and matched to cases by neighbourhood. Controls were identified using a reverse telephone directory system . We excluded potential controls who reported having diarrhoea within the 14 days preceding the date of illness onset to the matched case . The questionnaire for the case - control study was developed based on the findings from hypothesis generation including open - ended interviews and structured hypothesis - generating questionnaires . Over 40 exposures of interest included where the household purchased food, as well as consumption of spices and meats, and other foods reported with> 50% frequency during hypothesis - generating interviews . Cases were asked about exposures in the week preceding illness onset and controls were asked about the week preceding their interview . The rhode island department of health (ridoh) and the united states department of agriculture's (usda) food safety and inspection service (fsis) conducted a joint investigation at the manufacturing facilities of a company that produced italian - style meats . Salami product manufacturing processes were evaluated regarding the effectiveness of lethality steps and the potential for post - processing contamination . Food and drug administration (fda) collected finished product, red and black pepper applied post - lethality step, and environmental samples . As part of the environmental risk assessment at the company that produced italian - style meats, environmental testing was conducted from the receiving areas to the finished product storage areas . Ridoh also conducted standard plate count testing for pepper to determine the effectiveness of supplier treatment and for pepper used on ready - to - eat (rte) salami products . The pepper, salami products, and environmental samples were cultured at private, state public health and agriculture laboratories, fsis, and fda laboratories . Fda collected and analysed 153 composite pepper samples, which represented more than 4000 individual samples of black and red pepper . State public health laboratories serotyped human salmonella isolates and subtyped human s. montevideo isolates by pfge using standard pulsenet methods [8, 9]. We entered data into a microsoft access database and conducted analyses using sas v. 9.2 software (sas institute inc . Standard univariate and multivariate methods were used to examine the relationship between exposure and illness . In august 2009, an increase in the number of cases of s. montevideo with an indistinguishable pfge pattern was identified . Cases were geographically dispersed and the number of cases was not far above the 1015 cases per month expected for this common pfge pattern, so the cluster was monitored for additional cases . By november 2009, the cluster had increased to 102 cases in 30 states . At this time a total of 272 cases were identified from 44 states and the district of columbia (fig . 1); illness onset dates ranged from 1 july 2009 to 14 april 2010 (fig . The median age of patients was 37 years (range <193 years); 53% (144/272) were female . Twenty - six percent (52/203) were hospitalized; no deaths were reported . 1.persons infected with the outbreak strain of salmonella montevideo, usa, by state, 1 july 2009 to 14 april 2010 (n=272). Fig . 2.infection with the outbreak strain of salmonella montevideo in the usa, 20092010, according to week of illness onset (n=272). * reported and estimated . Persons infected with the outbreak strain of salmonella montevideo, usa, by state, 1 july 2009 to 14 april 2010 (n=272). Infection with the outbreak strain of salmonella montevideo in the usa, 20092010, according to week of illness onset (n=272). * reported and estimated . During 30 november 2009 to 16 december 2009, we completed 53 hypothesis - generating questionnaires from case - patients in 18 states . No food or other exposures were reported in an unusual frequency when compared with data available from the 20062007 foodnet population study . Although these questionnaires did not identify a clear hypothesis for a possible source of the outbreak, they helped identify three subclusters of cases . It was reported that fruit, delicatessen meat and cheese trays were served at the wedding . The second was a hunting group from south carolina that travelled to south dakota and included one confirmed case - patient and two probable case - patients . The case - patients reported consuming only milk, bread, alcohol, and homemade pork barbeque seasoned with crushed red pepper flakes during the trip . The third consisted of several cases in california that reported eating pork carnitas and salsa with various spices purchased at a common ethnic grocery store . During 16 december 2009 to 14 january 2010, we conducted 16 open - ended interviews of case - patients from eight states . Twelve (75%) case - patients reported consumption of italian - style meats, nine (56%) reported eating salami, and nine (56%) reported shopping at national warehouse store chain x. during 16 december 2009 to 14 january 2010, wadoh epidemiologists collected information from seven case - patients from two states regarding specific food items purchased at national warehouse chain x using information obtained from membership purchase records . Five of seven case - patients purchased a common company a rte salami variety package before illness onset . Using membership cards collected over the course of investigation, 19 ill persons were identified who purchased company a salami products before illness began: 16 persons purchased a salami variety package and three persons purchased a salami delicatessen tray . State and local health departments and cdc conducted a case - control study from 16 to 20 january 2010 . Case - patients were significantly more likely than controls to report consumption of salami [matched odds ratio (mor) 85, 95% ci 21759] (table 1). Consumption of any italian - style meat including salami, capocollo, calabrese, or sopressata was significantly associated with illness (mor 45, 95% ci 15183) (table 1). Case - patients were also significantly more likely than controls to report consumption of capocollo (mor 168, 95% ci 28), sopressata (mor 96, 95% ci 14), and prosciutto (mor 139, 95% ci 22) (table 1). However, when the cases reporting consumption of salami were removed from the analysis, capocollo, sopressata, and prosciutto consumption was no longer significant . Freshly ground black pepper added to foods was not associated with illness (mor 14, 95% ci 0544). Table 1.number and percentage of case - patients and controls reporting exposures in study of outbreak of salmonella montevideo infections, by type of exposure, usa, 1 july 2009 to 14 april 2010exposurecase - patients (n=43)controls (n=43)mor95% cino.%no.%salami2255061548520759capocollo1339400016828calabrese2560002402sopressata71940009614proscuitto1128200013922any italian - style meat2558192194515183freshly ground black pepper2054117436140544mor, matched odds ratio; ci, confidence interval. states include: arizona, california, colorado, connecticut, idaho, illinois, kansas, massachusetts, minnesota, north carolina, north dakota, new hampshire, new jersey, new york, ohio, oklahoma, oregon, south carolina, texas, washington.includes salami, capocollo, calabrese, sopressata, and prosciutto . Number and percentage of case - patients and controls reporting exposures in study of outbreak of salmonella montevideo infections, by type of exposure, usa, 1 july 2009 to 14 april 2010 * mor, matched odds ratio; ci, confidence interval . States include: arizona, california, colorado, connecticut, idaho, illinois, kansas, massachusetts, minnesota, north carolina, north dakota, new hampshire, new jersey, new york, ohio, oklahoma, oregon, south carolina, texas, washington . Product tracebacks based on warehouse store membership card information and purchase information of company a products by ill persons at other retail locations revealed that the salami products associated with illness were produced in three different rhode island establishments owned and operated by company a. prior inspections found no critical violations at the facilities . The salami products were fermented and cured (the critical control point for pathogen reduction) and some products were rolled in hot pork fat and coated with black or red pepper . No additional lethality steps were performed after the pepper was applied . During the inspection, company a used untreated pepper in raw products that were going to go through a curing process . They use the treated pepper on products post - processing . The treated and untreated pepper the supplier to company a reportedly tested the pepper for pathogens, but no further testing of the pepper was done by company a. company a did test salami at the end of fermentation (prior to the application of pepper) and none found salmonella . On 26 january 2010 a private laboratory identified salmonella serotype senftenberg with pfge pattern jmpx01.0004 from an unopened company a sliced salami product purchased at retail . Wadoh subsequently tested a culture from this product provided by the private laboratory and identified the outbreak strain of s. montevideo in addition to s. senftenberg . In total, either the outbreak strain or s. senftenberg was isolated from six open company a salami products collected from ill persons' homes and four intact / unopened retail products . Between 1 july 2009 and 14 april 2010, pulsenet identified 11 persons who had s. senftenberg infections with pfge pattern jmpx01.0004 . Public health officials interviewed nine of 11 ill persons with this strain of s. senftenberg; two reported purchasing a recalled company a salami product during the week before illness onset . The outbreak strain was identified in three sealed boxes of black pepper and three sealed boxes of red pepper collected at company a. s. montevideo isolates from black and red pepper were also found to have a similar genetic identity to isolates from case - patients based on molecular next - generation sequencing techniques . S. bareily and s. newport were identified in an intact container of red pepper at company a. s. senftenberg and s. virchow were also identified in two sealed boxes of black pepper and one box of red pepper at two different consignees of a spice company that supplied company a. pfge patterns of isolates from these salmonella serotypes were not found to match any human cases reported to pulsenet . Ridoh and fda collected samples of black and red pepper from company a and tested for standard plate counts . Standard plate counts ranged between 4400 and 150 000 colony - forming units per gram (c.f.u./g) in seven sealed containers of untreated black pepper samples and between 8500 and 2 000 000 c.f.u./g were isolated from eight sealed containers of untreated red pepper . Treated black and red pepper samples were also tested for standard plate counts . Standard plate counts ranged between 3000 and 83 000 c.f.u./g in sealed containers of irradiated black pepper and 3000 c.f.u./g were isolated from one sealed container of irradiated ground red pepper . According to the hpa guidelines for assessing the microbiological safety of ready - to - eat foods, pepper is often contaminated with at least 10 c.f.u / g of bacillus spp . . An environmental swab collected from a floor drain in the fermentation room of a company a production facility yielded s. montevideo indistinguishable from the outbreak strain . Fda initiated investigations at three pepper suppliers of company a: spice company b, spice company c, and spice company d, as well as several consignees of these spice companies . A sample from a consignee of spice company b, was positive for s. virchow . A sample collected by fda from a market in rhode island was positive for s. senftenberg with a pfge pattern indistinguishable from the pattern found in other company a products; and sourced to a consignee of spice company d. the market was using the pepper in the manufacturing of sopressata . The sopressata was tested at ridoh and found to be positive for s. typhimurium . As a result of finding salmonella in both the pepper and the finished product, pfge patterns of isolates from these s. virchow and s. typhimurium serotypes were not found to match any human cases reported to pulsenet . Pepper tracebacks revealed the black and red pepper originated from three asian countries . A common source in the distribution path from production to the company a facility was not identified between the black and red pepper . On 23 january 2010, company a recalled [12, 13] 590 000 kg of rte varieties of salami products . Based on additional findings of salmonella in company a products, the recall was expanded on 31 january, adding 7700 kg of salami products and on 16 february, adding 52 000 kg of salami products . On 25 february 2010, spice company b recalled 24 000 kg of crushed red pepper and on 5 march 2010 spice company d voluntarily recalled two lots of black pepper totalling nearly 25 000 kg . On 5 march 2010 the rhode island market that used pepper in the manufacturing of sopressata issued a recall of its products . The number of cases with the outbreak strain identified by pulsenet returned to the expected baseline number of cases by early 2010 . This nationwide outbreak of s. montevideo infections was associated with rte salami products made with contaminated black and red pepper added after the critical control point for pathogen reduction . This outbreak highlights the importance of preventing raw ingredient contamination and the potential for spices, such as pepper, contaminating rte products . This outbreak also reveals the importance of manufacturer verification of the safety of ingredients used in rte foods after the critical control point for pathogen reduction . The implicated pepper had reportedly been treated and tested by suppliers prior to use . However, high standard plate counts and salmonella were identified from intact containers of treated and untreated pepper . The starting bacterial load for some of these products was extremely high indicating that treatment was insufficient . Use of membership card information provided critical clues to identify the source in this investigation . State and federal regulatory partners identified production lots of black pepper and red pepper used to produce the contaminated salami products using purchase information gathered from these cards . Membership cards can provide significant information to identify contaminated foods quickly and help verify potential exposures in patients . This technique has been effective in providing additional epidemiological evidence in other outbreak investigations [1417]. After the source of the outbreak was identified and the case - patients from the wedding and hunting group subclusters were re - interviewed, state and local health departments were able to confirm from shopper card information that the wedding in arizona was catered with company a italian - style meats and members of the hunting group reported purchasing an italian - style delicatessen meat tray . When available, membership card information should be considered for use in future foodborne disease outbreak investigations . Twelve spice - associated salmonella outbreaks have been reported in nine countries between 1973 and 2009 with 1783 illnesses, at least 124 hospitalizations, and one death . European countries reported several salmonella outbreaks associated with salami and other fermented sausage products [1923]. However, these outbreaks resulted from insufficient curing time, low water activity, and high ph of the salami, allowing salmonella to survive [1923]. The source of this outbreak was contaminated pepper coated on salami products after the lethality step . Although spices are sometimes known to harbour various moulds, fungi, and bacteria, relatively few reports have documented spices as the cause of human illness . This may be because of the difficulty of identifying a source of an ingredient - driven outbreak . A variety of effective methods exist to decontaminate spices including steam, ethylene oxide, propylene oxide treatments, and irradiation . However, companies are not currently required to treat spices and manufacturers are not required to use treated spices in their products . These treatment methods have increased in importance given the frequent use of spices in rte foods and the potential for contaminated spices to cause widespread outbreaks . Manufacturer validation of the effectiveness of these treatments along with appropriate post - treatment sampling to verify the process is also critical . Any finding of salmonella is a violation of the federal food, drug and cosmetic act . We detected this outbreak through routine serotype - based surveillance enhanced by pfge reported to pulsenet . A case - control study identified an exposure significantly associated with illness, but the investigation using warehouse store membership cards revealed specific product and manufacturer information . Many of the case - patients could not remember the exact brand of salami that they had eaten or purchased and the membership card information was critical in identifying this information during the investigation . Information from membership cards should be used in future foodborne outbreak investigations to help identify suspect food items . Each member of the household may have a different membership card number making it difficult to receive all of the purchase records . For example, case - patients may purchase foods from different stores that do not have membership cards, many food products are consumed outside the household and not recorded on a card, and the central database of a store does not always contain data on all foods sold such as foods purchased from a salad bar . Cases were interviewed about food exposures before illness onset and controls were asked about the period prior to interview . Without illness to clearly delineate a time period, controls might have more difficulty recalling the timing of exposures . However, when compared to the foodnet population survey atlas of exposures, there was not a difference in the amount of salami or prepackaged delicatessen meats by month reported by healthy people . As a result of this investigation, the fda has increased sampling and surveillance of imported pepper spices and continued previously initiated discussions with the american spice trade association regarding preventive controls to safeguard against contaminated spices entering commerce . Manufacturers should implement supplier verification and preventive control programmes in an effort to eliminate contamination of their products . Foodborne disease outbreak investigations should seek to identify a root cause to help prevent future outbreaks . Without ingredient testing and a root cause investigation in this investigation, a recall would not have occurred, the plant would have gone back into production using contaminated pepper, and illnesses could have continued.
Type 1 diabetes mellitus (t1 dm) is one of the most common chronic diseases in childhood and results from autoimmune destruction of pancreatic -cells, leading to insulin deficiency . T1 dm is thought to originate through a combination of genetic and unknown environmental factors, of which environmental factors remain poorly defined . Newfoundland and labrador (nl), canada, is recognized as having one of the highest rates of t1 dm worldwide . A study on hospitalizations of children in nl reported an increase in diabetes - related hospitalizations among children aged 019 years . T1 dm is a significant disease in nl with its associated acute and chronic complications as well as the economic costs to both families and the health care system . Identification of potential perinatal environmental risk factors is examined in this study to try and elucidate potential reasons of why the disease is so common in this region of north america . This study was a case control design involving the linkage of data extracted from the newfoundland and labrador diabetes database (nldd) with the live birth system (lbs). The nldd is maintained by the janeway pediatric research unit at the janeway child health care centre (jchcc) in st . The nldd includes data for the majority of cases of t1 dm diagnosed in nl from 1987 to present . Children are included in the database as part of a prospective provincial study on the epidemiology of t1 dm in nl . They have a confirmed diagnosis of t1 dm according to canadian diabetes association (cda) criteria . The lbs is maintained by the newfoundland and labrador centre for health information (nlchi). Data for this system are acquired from live birth notification forms that are completed in all provincial health care facilities . The forms are provided to nlchi by the vital statistics division, service nl, and contain clinical and demographic data for all births (resident and nonresident) in the province . Cases included children born in nl from 1992 onwards which have been diagnosed with t1 dm before 15 years of age . Children with type 2 diabetes, maturity - onset diabetes of youth, transient hyperglycemia, and diabetes caused by chemotherapy or cystic fibrosis are excluded from the nldd and thus were not included in the study . Children born prior to 1992 were not included in the study because there are no electronic birth notification data available before this date . A unique identifier, such as the provincial health insurance plan number, was not available for all children in the nldd . As a result case subjects were linked to the lbs using child's date of birth and mother's maiden name . Of the 301 cases in the nldd, 23 were excluded because they were born out of province . Of the remaining 278, linkage was possible for all but six children resulting in a total of 266 cases included in the study . Three control subjects (n = 798) were selected for each case matched on year of birth, sex, and health authority of mother's residence at time of delivery . Power analysis was performed to determine whether this sample size would be sufficient to detect statistical significant associations between t1 dm and the risk factors of interest . The power analysis was conducted considering an overall rate of birth by c - section in nl as 30.9%, in order to achieve a power of 80% with a desired odds ratio of 1.5 . Using the method described by kelsey et al . The power analysis confirmed that a sample size of 266 cases and 798 controls is sufficient to detect statistically significant relationships between t1 dm and delivery by c - section . Cases and controls were grouped into two gestational age categories: preterm and term / postterm . Birth weight in grams was used to classify cases and controls as high birth weight (> 4,000 grams) or not (4,000 grams). Cases and controls were also classified as small / appropriate - for - gestational - age or large - for - gestational - age using the method described by kramer et al . . Cases and controls were grouped according to parity or birth order as either 1 and 2 or more . Mother's marital status was categorized as married, single, separated, widowed, or divorced . Mother's education level was classified into three categories: not graduated high school, graduated high school, and education beyond high school . Demographic and clinical factors of mothers, including age, marital status, education, place of residence, parity (number of live born children delivered), and complications of pregnancy, were included . Cases and controls were analyzed by sex, place of residence, age of onset, length of gestation, type of delivery, birth weight, size for gestational age, and birth order . Chi - square tests were used to predict diabetes status on the basis of the independent variables . Conditional multiple logistic regression was used to assess the relationship between t1 dm risk and the variables of interest . The first model contained birth weight, gestational age, parity, delivery method, mother's marital status, mother's education level, mother's age, maternal hypertension, and mother's t1 dm status . The second model incorporated all variables in the first model with the exception of birth weight and gestational age which were replaced with size - for - gestational - age . Birth weight and gestational age were not included in the same model as size - for - gestational - age as they are components of this variable . The statistical package for the social sciences (spss) 17.0 this study received approvals from the human investigation committee of memorial university, from each of the hospital boards and the secondary uses committee of the newfoundland and labrador centre for health information (nlchi) prior to commencement . The percentages of male and female cases were similar (50.8 and 49.2, resp . ). Table 1 also demonstrates the age distribution of cases as well as their age of diagnosis . There were more males than females diagnosed in the 04 age group; however, this finding was not statistically significant . Table 2 presents maternal and perinatal characteristics of the study population . A higher percentage of cases than controls were born pre - term (9.8% versus 6.8%, resp . ). While there was no significant difference observed between birth weight of the cases and controls, there was a significant difference observed for size - for - gestational - age with a higher percentage of cases than controls born large - for - gestational - age (18.2% versus 12.8%, resp . It was more common for cases to be delivered by c - section than controls (30.8% versus 22.1%, p = 0.009). T1 dm was more common among first or second born cases compared to those born third or higher (p = 0.022). Table 3 presents the results of the conditional logistic regression models . In the model which included birth weight and gestational age, delivery by c children delivered by c - section were 1.41 times as likely to develop t1 dm (hazard ratio (hr) 1.41, p = 0.015). In the second model, which included size - for - gestational - age, c - section delivery was not associated with increased risk of t1 dm (hr 1.3, p = 0.076). Both parity and size - for - gestational - age were found to be significant risk factors for t1 dm from chi - square analysis (table 2); these factors did not remain statistically significant in the conditional logistic regression models . Findings of this study indicate that c - section delivery was a significant risk factor for t1 dm in children aged 015 years . This finding is in line with a recent meta - analysis of 20 studies which found that the combined effect of c - section delivery was 1.23 (95% ci 1.151.32). Theories of why this may be associated with the development of t1 dm in offspring includes the involvement of the role of gut bacteria in the development of the immune system . Studies have shown a difference between the compositions of gut microbiota in vaginally delivered children and those delivered by c - section . Children delivered by c - section may be primarily exposed to bacteria in the hospital and not maternal bacteria, hence the increased risk of t1 dm may be linked to a different composition of gut flora . Another possible explanation is related to the hygiene hypothesis which proposes that the risk of diabetes may be increased when children are not exposed to infections in early life . Children delivered by c - section have decreased exposures to infections compared to children born vaginally and, in turn, have increased risk for diabetes . Another theory suggests that the observed increased risk of diabetes after c - section may be related to perinatal stress . Nl has a high rate of birth by c - section as compared to other regions in canada . The provincial rate of births by c - section was 30.9% in 2005 - 2006 versus the canadian rate of 26.3% . The rates of c - section have increased in nl to 33% in 2010 . In the present study, maternal age at time of birth was not found to be significantly associated with risk of t1 dm in offspring; other studies have found significant relationships between mother's age and t1 dm risk . A recent meta - analysis of 37 studies found that the odds of t1 dm increased by 10% for children whose mothers were over 35 years of age at time of birth (or = 1.10 95% ci 1.01, 1.20; p = 0.03). Conversely, a matched case - control study of 196 cases in the united kingdom found that mothers of control children were older than mothers of cases (or = 0.900 95% ci 0.854, 0.948; p <0.001). For other maternal factors, such as education level and marital status, there were no associations found which is consistent with other similar studies [14, 15]. The present study also did not find any associations between maternal hypertension and risk of t1 dm in offspring . Other studies have found an increased risk of t1 dm in offspring with maternal history of t1 dm [13, 16, 17]; however, these studies also included information on paternal history of t1 dm . A 2009 study by algert and colleagues did not find an association between maternal t1 dm and risk of t1 dm in children . Similar to the present study, the study by algert et al . Did not contain information on paternal t1 dm . There was no significant relationship between parity and t1 dm risk found in the current study . This is different than a western australia population based cohort study of 835 cases of t1 dm diagnosed by the age of 15 that found a significant decrease in t1 dm with increasing birth order . Birth weight and gestational age were not found to be associated with risk of t1 dm in the present study; however, chi - squared analysis revealed a significant difference between t1 dm and size - for - gestational - age with a higher percentage of cases than controls born large - for - gestational - age, but this was no longer significant in the conditional logistic regression models . Our findings do not support the findings of a meta - analysis of 11 studies examining birth weight which found that a birth weight greater than 4,000 grams was associated with an increased odds of t1 dm (or = 1.17 95% ci 1.09, 1.26; p <0.05). Findings related to the association between gestational age and t1 dm appear to be mixed . A case - control study conducted in austria found that babies born at 3439 weeks had a significantly higher risk for t1 dm compared to those born before 33 or after 40 weeks . However, a study by cardwell et al . Found that children born after 40 weeks gestation had a significantly lower risk of t1 dm than children born prior to 40 weeks . While size - for - gestational - age has not been extensively studied, some studies have found significant associations . A cohort study of 272 children in new south wales, australia, with t1 dm found that children who were small - for - gestational - age had a significantly decreased risk of t1 dm compared to children born appropriate - for - gestational age . The findings of this study should be considered in the context of its strengths and weaknesses . An important strength is that the use of a record linkage case - control study design eliminates recall bias that is apparent in cross - sectional study designs . Secondly, data contained in the lbs were collected at time of birth by healthcare professionals, and the nldd data were collected from physician charts at time of t1 dm diagnosis which contributes to the reliability of the data . A limitation of this study is that there was very little information available pertaining to fathers as the majority of the information collected at the time of birth for the lbs is related to the mother and child . This study identified c - section as a significant risk factor for the development of t1 dm among children aged 015 years in nl, a region with very high rates of t1 dm . Findings may have an impact on health practice, health care planning and future research related to t1 dm among children.
Gallbladder cancers (gbcs) are highly aggressive cancers with the highest mortality rate among gastrointestinal cancers . The highest prevalence rates of gbc have been observed in american indian, chilean, and japanese women [1, 2]. Early diagnosis of gbc is generally impossible due to a lack of specific signs or symptoms . Over 90% of gbc patients are diagnosed at an advanced, inoperable stage with serious invasion and metastasis to other organs . A majority of gbcs encountered in the clinic are adenocarcinomas (ac, 9095%), while rare subtypes such as squamous cell / adenosquamous carcinoma (sc / asc), mucinous carcinoma, signet ring cell carcinoma, and undifferentiated carcinoma are hardly ever encountered [2, 5]. At present, cholecystectomy is only viable for early stage gbc [68], while patients with unresectable or metastatic gbc have extremely poor prognosis and few treatment options . Generally, palliative chemotherapy and radiation therapy offer limited benefits to patients with gbc . Therefore, there is an urgent need to identify biological markers for the diagnosis, prognosis, and target therapy of gallbladder cancers . Numerous studies have observed elevated levels of oxidative stress during tumor growth, which is characterized by an increase in reactive oxygen species (ros). Ros serve as secondary messenger molecules to increase tumor cell proliferation, genetic mutations, and instability, which lead to subsequent invasion, angiogenesis, and drug resistance in cancer cells . Most importantly, cancer cells can develop mechanisms to evade high ros - induced apoptosis [10, 11]. The elevation of oxidative stress levels in tumor cells is generally thought to be associated with two factors: the increased generation of ros and the decreased capacity of the cell to eliminate ros . Enhanced release of o2 and/or h2o2 from the mitochondria and activation of nadph oxidase (nox) systems increase cellular ros levels [13, 14]. The increase in ros can also be caused by the suppression of antioxidant enzymes, such as mnsod and gpx . Thus, the differential expression of antioxidant enzymes in cancer can serve as biomarkers of tumor initiation and/or progression . Tumor suppressive roles of the superoxide dismutase (sod) family of proteins have been observed in a variety of cancers . Glutathione peroxidase (gpx) has been revealed to modulate the tumor suppressive effect of mnsod in various tumor cells . A number of studies have demonstrated that gpx3 expression is downregulated in a variety of cancers . For example, gpx3 is strongly downregulated in prostate cancer, thyroid cancer, colorectal cancer, gastric cancer, and breast cancer [15, 16]. Intriguingly, a previous study has revealed that gpx3 expression is higher in clear cell epithelial ovarian carcinoma tissues compared to control tissues . Since gpx3 is always highly expressed in healthy tissues, it has been suggested to be a tumor suppressor in many tumors . Aldehyde dehydrogenases (aldhs) are a family of enzymes that metabolize both endogenous and exogenous aldehydes to carboxylic acids as well as other reactive compounds . For example, aldh1a1 and aldh3a1 function as ocular antioxidants that play unique roles in the defense of the eye against uv radiation and in the downstream effects of oxidative stress . Some aldh isoforms function in retinoic acid (ra) signaling via ra production by oxidizing all - trans - retinal and 9-cis - retinal . Recently, aldh1 activity has been employed as a marker of stem - like cells in many cancers, such as cervical and breast cancer . Moreover, aldh1 activity was demonstrated to be significantly higher in metastatic breast tumor cells that escaped chemotherapy . Furthermore, high activity of aldh1 is associated with poor prognosis in breast, bladder, and prostate cancer patients [24, 25]. However, most aldh prognostic data is based on using aldh1a1 expression levels as an indicator for cancer patients' outcome [26, 27]. These studies also yielded varied results, and the correlation of aldh1a1 with prognosis may be dependent on cancer type . In contrast, aldh1a3, but not aldh1a1, has been shown to correlate consistently with tumor grade and metastasis of breast cancer patients, suggesting that aldh1a3 may be a better marker in some tumors than aldh1a1 . The association of other aldh subtypes with tumor progression and prognosis has not been established . Our preliminary study using microarray assay showed a 24-fold increase in aldh1a3 level and 23-fold decrease in gpx3 in gbc - sd cells compared to normal gbc epithelial cells (data not shown). In this study, the expression of aldh1a3 and gpx3 in surgically resected specimens, including ac and sc / asc, was examined by immunohistochemistry . The correlation of aldh1a3 and gpx3 expressions with the clinicopathological characteristics and prognosis of ac and sc / asc was comparatively evaluated . A total of 46 squamous cell / adenosquamous carcinomas (sc / asc) from gbc patients that underwent surgical resection or biopsy were collected from january 1995 to december 2009 . The percentage of sc / asc in various gallbladder cancers is 4.34% (46/1060 gbcs). 80 adenocarcinomas (ac) samples for patients that underwent surgical resection or biopsy were collected from january 2005 to december 2009 . A gallbladder cancer sample was diagnosed as sc when most malignant cells are squamous cells and less than 10% are adenocarcinoma cells . A gallbladder cancer sample was diagnosed as asc when the tumor contains both squamous cells and adenocarcinoma cells, but the tumor must contain at least 10% adenocarcinoma or squamous cell carcinoma cells . Among the 46 sc / asc patients, 19 patients were male and 27 patients were female with an age variation from 35 to 82 (55.8 9.6) years . Among the 80 ac patients, 26 patients were male and 54 patients were female with an age variation from 33 to 80 (53.8 9.9) years . The differentiation (refer to squamous cells) of the 46 sc / ascs includes 16 well - differentiated, 24 moderately differentiated, and 6 poorly differentiated carcinomas . Among the 80 acs, 27 were well differentiated, 25 were moderately differentiated, and 28 were poorly differentiated . Among the 46 sc / asc patients, invasion of gallbladder, surrounding tissues and organs, was found in 30 patients, while 29 patients had regional lymph node metastasis . Among the 80 ac patients, invasion of gallbladder surrounding tissues and organs was found in 49 patients, while 50 patients had regional lymph node metastasis . Surgery included radical resection for 14 sc / ascs and 26 acs, palliative surgery for 18 sc / ascs and 28 acs, and no operation for 14 sc / ascs and 26 acs with only biopsy . The 2-year survival information of 80 ac and 46 sc / asc patients was obtained through letters and phone calls . This study was preapproved by the ethics committee for human research, central south university . Four - micrometer - thick sections were cut from routinely paraffin - embedded tissues of ac and sc / asc . Staining was conducted with the peroxidase - based envision detection kit (dako laboratories, ca, usa) by following the user manual and the rabbit anti - aldh1a3 (abgent company, california, usa) and rabbit anti - gpx3 (novus biologicals, littleton, usa) antibodies which specifically stain human aldh1a3 and human gpx3 . Briefly, the sections were deparaffinized and then incubated with 3% h2o2 for 15 minutes . After being soaked with phosphate - buffered saline (pbs) for 3 5 minutes, the sections were incubated with primary antibody for 1 hr at room temperature . After rinsing sections with pbs for 3 times, solution a was added, and the sections were incubated for 30 minutes . The substrate dab was added followed by hematoxylin counterstaining . The slides were dehydrated with different concentrations (70%100%) of alcohol and soaked in xylene for 3 5 minutes . The positive control was the positive biopsy provided by beijing zhongshan biotechnology company (beijing, china), while the negative control was designed by replacing the primary antibody with 5% fetal bovine serum . By considering that heterogeneity of staining pattern exists, the percentage of positive cells was calculated from 500 tumor cells in 10 random fields . The staining strength was graded as previously described: a score of 1 was given for no positive staining or a uncertainly weak staining; a score of 2 was given for weak to moderate staining; a score of 3 was given for moderate to strong staining . For gpx3, cases with both positive cells 25% and scores 2 were considered positive . A few cases with 10% to 25% positive cells and 3 scores in staining cases with both positive cells 10% and scores 2 were considered positive . The slides were systematically scanned, and 7 - 8 representative digital images were acquired from each slide . Digital images were quantified by image analysis with adobe photoshop, version 7.0 (adobe systems, san jose, ca), and the extent of staining was assessed as the percentage of positively stained area per field under 100 magnification . Data was analyzed using the statistical package for the social sciences version 13.0 (spss 13.0). The interrelationship of aldh1a3 or gpx3 expressions with histological or clinical factors was analyzed using, fisher's exact test, or student's t - test . Kaplan - meier and time series tests (log - rank test) were used for univariate survival analysis . Cox proportional hazards model was used for multivariate analysis and to determine the 95% confidence interval . The percentage of cases with age older than 45 years and tumor mass> 3 cm was significantly higher in sc / ascs than in acs (p <0.05). The percentage of cases with poorly differentiated tumor was significantly lower in sc / ascs than in acs . No significant differences in other clinicopathological characteristics were observed between sc / asc and ac patients . Envision immunohistochemistry revealed that aldh1a3 and gpx3 positive reaction was mainly localized in the cytoplasm of tumor cells in sc / asc (figure 1) and ac (figure 2), but a small amount of inflammatory reaction cells and fibroblasts was also stained . Western blot with aldh1a3 antibody showed one main band of 56 kda and a weak band of 22 kda, while western blot with gpx3 antibody showed one band of 25 kda (data not shown). No significant difference in the percentage of cases with positive aldh1a3 and gpx3 expressions was observed between sc / asc and ac patients (data not shown). As shown in table 1 and figure 3(a), the percentage of cases with positive aldh1a3 expression was significantly higher in tumor tissues from sc / asc patients with high tnm stage and lymph node metastasis compared to tumor tissues from cases with low tnm stage and no lymph metastasis (p <0.05 or p <0.01). The gpx3 expression was significantly lower in cases with higher tnm stage, lymph node metastasis, and invasion (p <0.05 or p <0.01). Aldh1a3 and gpx3 exhibited no significant association with other clinicopathological characteristics of sc / asc . The percentage of cases with positive aldh1a3 expression and negative gpx3 expression in ac tumors was significantly higher in cases with poor differentiation, large tumor mass, high tnm stage with lymph node metastasis, and invasion to the gallbladder surrounding tissues and organs compared to the cases with well - differentiated tumor, small tumor mass, lower tnm stage, no lymph metastasis, and no invasion (p <0.05, p <0.01, or p <0.001) (table 1, figure 3(b)). Survival information of 46 sc / asc patients was obtained through letters and phone calls . The followup time was 2 years, and patients that survived longer than 2 years were included in the analysis as censored cases . Thirty - three of the 46 sc / asc patients survived less than 1 year and 13 patients survived over 1 year (4 cases survived longer than 2 years) with an average survival time of 10.07 0.78 months . Fifty - seven of the 80 ac patients survived less than 1 year and 23 patients survived over 1 year (9 cases survived longer than 2 years) with an average survival time of 10.34 0.63 months . The kaplan - meier survival analysis in sc / asc patients revealed that the differentiation, tumor size, tnm stage, lymph node metastasis, and invasion (p <the average survival time of aldh1a3 positive patients was significantly lower than patients with negative aldh1a3 expression (p = 0.005), but the average survival time of gpx3 positive patients was significantly higher than patients with negative gpx3 expression (p = 0.002) (table 2, figure 4). Cox multivariate analysis showed that the differentiation, tumor size (3 cm), tnm stage, invasion, and operative procedure as well as aldh1a3-positive expression or gpx3-negative expression were negatively correlated with postoperative survival, suggesting that aldh1a3 positivity is a risk factor and gpx3 has a strong impact on prognosis of scs / ascs (table 3). The kaplan - meier survival analysis in ac patients revealed that the differentiation, tumor size, tnm stage, lymph node metastasis, invasion, and operative procedure (p <0.001) were significantly associated with the average survival time (table 2). The average survival time of aldh1a3-positive ac patients was significantly lower than patients with negative aldh1a3 expression (p <0.001), but the average survival time of gpx3 positive patients was significantly higher than patients with negative gpx3 expression (p <0.001) (table 2, figure 5). Cox multivariate analysis showed that the differentiation, tumor size (3 cm), tnm stage, lymph node metastasis, invasion, and operative procedure as well as aldh1a3-positive expression or gpx3-negative expression were negatively correlated with poor survival of ac patients (table 3). The clinical and pathological characteristics of squamous cell / adenosquamous carcinomas (sc / asc) of the gallbladder have not yet been widely recognized because these subtypes are rather rare . Moreover, no study so far has systemically compared the clinical, pathological, and molecular differences between the rare sc / asc subtype and the common ac subtype of gallbladder cancers (gbc). In this study, no significant differences in differentiation, metastasis, invasion, tnm stage, and prognosis were observed between these two subtypes . The clinicopathological presentations of sc / asc did not seem to be different from ordinary ac though more sc / asc patients exhibited larger tumor mass (> 3 cm) than ac patients . This suggested that squamous differentiation is as aggressive as glandular differentiation in the gallbladder, which is inconsistent with previous observations [31, 32]. Importantly, we found no differences in aldh1a3 and gpx3 expressions between sc / asc and ac patients, suggesting that they may have similar biological characteristics . Numerous studies have observed an increase in oxidative stress during tumor growth, including gbc . Therefore, the expression of aldh1a3 and gpx3, two oxygen stress - related molecules, was investigated in this study . Previous studies have demonstrated that gpx3 is downregulated in a variety of tumors, such as prostate cancer, thyroid cancer, colorectal cancer, gastric cancer, and breast cancer [15, 16, 18]. However, the predictive role of gpx3 in the prognosis of these cancer patients has not been observed . Moreover, no molecular markers related to the aggressive biological characteristics or prognosis of sc / asc have currently been identified . In this study, we demonstrated that loss of gpx3 expression was significantly associated with metastasis, invasion, tnm stage, and poor prognosis in both sc / asc and ac patients . These observed correlations may possibly indicate that gpx3 is a tumor suppressor in gallbladder cancers . Increases in reactive oxygen species levels have been associated with tumor cell proliferation, genetic instability, and subsequent increases in invasion, angiogenesis, and drug resistance [9, 10]. Although this study provided no evidence on whether the expression and/or activity of other antioxidant enzymes were decreased and whether the suppression of these enzymes plays a key role in the increased ros levels in gbcs, the observed decrease in gpx3 expression suggested its involvement in oxidative stress . Aldehyde dehydrogenases are known to modulate several cell functions, including proliferation, differentiation, and survival, as well as cellular response to oxidative stress . In addition, cancer cell - acquired drug resistance has been demonstrated to be associated with the transcriptional activation of aldh1 expression . In contrast, aldh1a1 and aldh3a1 have been demonstrated to function as ocular antioxidants . In this study, positive aldh1a3 expression in ac patients was associated with poor differentiation, large tumor size, high tnm stage, lymph node metastasis, and invasion . In sc / asc patients, positive aldh1a3 expression was also associated with high tnm stage, invasion, and lymph node metastasis . This suggests that aldh1a3 in gbc plays a role in the proliferation, differentiation, and survival of tumor cells . In addition, both the ac and sc / asc patients with positive aldh1a3 expression exhibited poor survival . Recently, aldh1 activity has been employed successfully as a stem cell marker in various cancers . In addition, aldh activity detected in tumor cells may actually be an indication of stem cells, because they are resistant to various chemotherapeutic drugs and linked to poor prognosis [2225]. We acknowledge that although positive aldh1a3 expression or negative gpx3 expression significantly correlated the shorter survival in both sc / asc and ac patients, their regression coefficients or relative risk showed that the impact of positive aldh1a3 expression or negative gpx3 expression on survival was small when compared with differences in tumor size, invasion, and differentiation . Therefore, aldh1a3/gpx3 may be more effective as markers for proliferation and differentiation of gallbladder carcinoma than as tools for predicting overall survival . However, the real role of aldh1a3/gpx3 on tumor growth and metastasis of gallbladder cancer should be investigated further in animal models using inhibitory agents of aldh1, such as sirna and/or overexpression of gpx3 gene . The data from this study also raised questions on why do sc / asc and ac share very similar gene expression profiles for aldh1a3 and gpx3, and why the expression profiles of aldh1a3 and gpx3, both molecules associated with oxidative stress, are closely related to tnm staging and metastasis . In conclusion, the lowered expression of gpx3 and elevated expression of aldh1a3 in ac and sc / asc subtypes of gbc are important biological markers for the metastasis, invasion, and maybe prognosis of gallbladder cancer.
It is generally accepted that papillary thyroid carcinoma in children and adolescent population has a different clinical presentation and course than in adults . Initial diagnostic presentation in pediatric patients tends to be in more advanced stage, namely, the larger primary tumor, the higher incidence of primary tumor multicentricity and higher incidence of locoregional metastatic spread . More specifically, palpable neck lymphonodes are most commonly presenting sites in children (up to 90%). It is already demonstrated that primary thyroid tumor aggressiveness (as measured by nuclear atypia, tumor necrosis, and lymphovascular invasion) correlates with metastatic disease, independently from tumor size . Consequently, histological grade should be set as prognostically important component and included in any classification system of differentiated thyroid cancer . Despite these aggressive characteristics, specific prognosis in pediatric age is better than in adults . As a consequence, several recommendations have been made that propose less aggressive therapeutic approach (less than total thyroidectomy and no neck dissection). On the contrary, some researchers advise more aggressive therapy, including total thyroidectomy, neck lymphonode dissection, and postoperative radioiodine ablation [46]. However, recurrent disease and locoregional spread still present major clinical concern regarding optimal extent of the initial surgical therapy, in pediatric population specifically . Several major studies attempted to evaluate influence of patient and tumor characteristics, as well as treatment factors, but without clear recommendations regarding possible risk factors for developing recurrent disease [711]. In this paper, we show our retrospective clinical material in 16 patients up to 18 years of age, who underwent surgical therapy at our institution for diagnosis of thyroid papillary carcinoma . The aim of the presentation is to give insight into clinical and selected pathological characteristics (tumor size and aggressiveness, multicentricity, and locoregional spread) of the disease in pediatric age, assess their role in cancer risk, and identify possible critical parameters for developing metastatic disease . This study is a retrospective analysis of data from 16 pediatric patients with papillary thyroid cancer, ages from 1018 . All patients were operated upon at the department of ent head and neck surgery, university hospital sisters of charity, zagreb, croatia during the 28-year period (19802008). All patients underwent total thyroidectomy with or without neck dissection (paratracheal or some type of lateral neck dissection). 's neck dissection classification data from consensus statement on the classification and terminology of neck dissection . Medical data were collected from patient documentation (intraoperative reports, recurrences, fnab results, and additional thyroid diagnosis) and final pathology reports, as well as from the hospital registry for thyroid diseases were reviewed . Postoperatively, we followed diagnostic and therapeutic guidelines for differentiated thyroid cancer, issued by croatian thyroid society; postoperative diagnostic scintigraphy was performed with 13 mci i. high - risk patients were put to 100200 mci i ablation without l - t4 . Posttherapeutic whole body scintigraphy was performed 58 days after i. six to twelve months later, the patients were followed up with the exploration of neck ultrasound, ft4, tsh, tg, and tga (without l - t4) measurements and afterwards yearly . All patients were assigned to groups for variables of age (18 and comparison group> 18 years), gender, size (diameter) and pathological parameters of primary tumor aggressiveness, multicentricity, type of neck dissection performed, and presence of locoregional metastatic foci in neck . Grading of pathological aggressiveness, multicentricity, and locoregional spread is demonstrated in table 2 . We applied statistical analysis of differences with test and model of multivariate logistic regression for risk factors for metastatic locoregional spread . From the total of 699 cases with papillary thyroid cancers of all ages, 16 patients (2.3%) with age up to 18 years fulfilled inclusion criteria, 13 females, and 3 males . The average age at the moment of diagnosis was 16, with a range of 10 to 18 years . Majority of patients presented with palpable thyroid mass, 12/16 or 75%, and palpable neck node in 8/16, or 50% . There were only 4 microcarcinoma cases (25%), which is in sharp contrast to older population (45% microcarcinoma). Distribution of parameters of pathological aggressiveness, multicentricity, neck dissection, and metastatic spread and comparison by age group (18 and> 18 years) are shown in table 3 . Results showed that 60% of pediatric tumors were aggressive, according to our chosen criteria . Forty percent of tumors were multicentric, with foci in both lobes twice as often as in the single lobe (13% versus 27%). There were 77% positive metastatic tumors: paratracheal metastases in 23% and lateral neck metastases in 54% . Overall, no age differences were found for pathological aggressiveness (p = 0.19) and multicentricity (p = 0.89) even though younger group had significantly wider aggressiveness (34% versus 16%). Younger patients had significantly more neck dissections performed (p = 0.005) and more metastatic spread as well (p = 0.000). We had two cases of temporary early postoperative hypocalcaemia, which were successfully controlled by calcium carbonate and rocaltrol p. o. there was one case of permanent hypocalcaemia (recurrent case). There were two cases of disease recurrence, both locoregional: one patient with single neck recurrence (lateral neck metastatic foci after initial tt and paratracheal nd) and one patient with two recurrences (both in lateral neck regions after initial tt and paratracheal nd). Both recurrences were detected on follow up by neck palpation and ultrasonography with cytology, and pathologic tg; both cases were amenable to surgical therapy and were put to further radioiodine treatment . No patients with n0 neck developed recurrence . In all patients, postoperative and postablative average follow up was at least 5 years, and they were alive and free of disease . Multivariate analysis revealed the presence of multicentric foci in contralateral lobe and higher tumor aggressiveness (group iii) to be an independent risk factor for regional metastatic spread (or 3.119 and 2.591, resp . ). Male gender was identified as an additional risk factor (or 1.919), while older age was associated with lower risk for metastatic development (or 0.537). Different potential prognostic factors of papillary thyroid cancer have been revealed so far, most importantly patient age, tumor grade, and extension (extrathyroid invasion, distant metastasis, and less frequently regional). Papillary cancer characteristics and behavior in children and adolescent population is subject of several controversies, mainly about the most appropriate surgical therapy, the use of postoperative iodine ablation and tsh suppression, as well as follow - up modalities [1, 13]. It is well demonstrated that clinical course of this disease in children and adolescent population is significantly different: the worse initial clinical presentation in children and, paradoxically, the better disease prognosis than in adult population [14, 15]. Children tend to present with larger primary tumors, greater incidence of neck lymphonode metastasis, and distant metastasis as well . Possible explanations are more common occurrence of well - differentiated forms of tumor and more effective response to postoperative tsh suppression with thyroid hormone . In our series most aggressive cases came in the form of a wider tumor aggressiveness, that is, thyroid capsule invasion, penetration of adjacent thyroid tissues, and/or perivascular / perineural spread . Additionally, mean tumor size in younger age group was significantly larger than in adult population . The role of tumor size was commented recently in the review of sherman, who emphasized that smaller size of thyroid gland in children can lead to earlier thyroid and extrathyroid spread of disease . Papillary thyroid cancer multicentricity is a well - described feature of this tumor, with estimated frequency range from 22% to 49% . There are major disagreements about the importance of papillary cancer multicentricity; its etiology and clinical significance are not yet fully understood [6, 1820]. As a consequence, many authors propose total thyroidectomy as an adequate surgical approach, claiming that more extensive operation decreases the likelihood of recurrence . Thus, multicentricity, when it occurred, was bilateral in more than two - thirds of multicentric cases . Further, on multivariate analysis multicentricity was found to be the most important risk factor for development of metastatic spread (or 3.119). Interpretations of these findings strongly signify the need for an implementation of more aggressive surgery of the primary tumor, incorporating complete removal of the contralateral thyroid lobe in the same act as thyroidectomy . Locoregional metastatic spread is identified as one of the most important risk factors preceding distant dissemination . Neck metastases occur in 15%60% and with meticulous search can be found in up to 90% of cases [22, 23]. However, even for distant spread, chances of long - term survival are significant, particularly in younger age group ., there were 77% positive metastatic tumors: paratracheal metastases in 23%, and lateral neck metastases in 54% . It has been demonstrated that metastatic disease, locoregional and distant, seems to be most important prognostic factor for the good response to therapy . Data from present study clearly demonstrate the significance of detailed preoperative neck examination and careful intraoperative exploration, with special emphasis on lateral neck regions . Presently, total or less commonly subtotal thyroidectomy is considered optimal surgical therapy for papillary thyroid cancer in all preoperatively diagnosed cases [18, 26]. Conducted studies showed that more extensive operation (total thyroidectomy) usually leads to significant reduction of recurrence [9, 13, 27]. A more conservative surgical approach (lobectomy) is advocated by some authors as well, emphasizing less risk of surgical and postoperative morbidity (temporary or permanent hypoparathyroidism and recurrent laryngeal nerve injury) [11, 28, 29]. Thyroid cancer recurrence is more common in extremes of age, most notably in patients less than 20 years of age, as well as older than 60 [4, 30]. Generally, studies performed on all age populations revealed higher chances for recurrence in procedures including less than total thyroidectomy . This higher aggressiveness of thyroid cancer demonstrated in younger population does not necessarily bear higher risk for fatal outcome, which is more common in older population [14, 15]. Our both recurrence cases had a large primary tumor in the thyroid gland; one had aggressive characteristics (thyroid capsule invasion and perivascular, perilymphatic spread), and both had initially neck metastatic disease . Regarding intraoperative and early postoperative complications rate, in our material, we have not found significant differences in perioperative complication rates in children versus older patients, with extent of surgery being the same as for adults . In recurrent cases, reoperative surgery, which consisted of lateral neck dissection, was performed without any complications . However, in this cohort of patients, we advise that strong emphasis should be put on minimizing morbidity related issues, bearing in mind excellent long - term disease prognosis . In conclusion, in this study, we investigated parameters of papillary thyroid cancer aggressiveness, multicentricity, and locoregional metastatic spread in children / adolescent population . Overall, papillary thyroid cancer demonstrated aggressiveness in 60%, intraglandular dissemination in 40%, and metastasized locoregionally in 77% of cases . By observing clinicopathological parameters and their distribution across selected groups multicentric foci in both thyroid lobes and wider tumor aggressiveness were identified as a risk factor for metastatic development . We support the need for total thyroidectomy and meticulous intraoperative exploration of the thyroid bed and lateral neck, with surgical extirpation of all potential microscopic disease foci . For positive regional metastatic disease, standard paratracheal and/or modified radical neck dissection is obligatory part of surgery, in the same act as total thyroidectomy, keeping in mind that neck lymphonodes are primary sites of locoregional recurrence . With meticulous attention to technical aspects of surgery
Much research relating to the control of air conditioning systems for living environments and office spaces has been performed but that on outdoor spaces is incomplete . It has been shown that, in addition to physical and physiological environmental factors, psychological environmental stimuli are also important for the determination of thermal comfort . In contrast to indoor spaces, it is not just the thermal environment stimuli but also the environmental complex consisting of visual and auditory stimuli that has an influence on comfort . Adaptation to the thermal environment according to cultural background, experience of being in hot / cold thermal environments and the like, sense of expectation of the thermal environment, behavioural thermal regulation, and the effect of thermal environment history such as the time spent exposed to it are strongly apparent [18]. Although thermal comfort is the subject of research, it is treated as nonspecific and comprehensive rather than particular . In the case of restricting the responses to thermal stimuli in instructions for the experiment and not having subjects respond on thermal sense, a nonspecific evaluation is a thermal evaluation but is also the evaluation of a comprehensive impression of space by environmental stimuli other than thermal stimuli, such as visual or auditory stimuli [1014]. In contrast, a specific evaluation is when the test subjects are made to focus on and evaluate thermal stimuli alone as instructed by the research staff . Research specifically on thermal factors is rare in comparison with research that deals with a thermally nonspecific comprehensive sense of comfort [1519]. Such research is carried out with the objective of finding the comfort zone of an outdoor space . The hot and cold that humans sense is typically investigated by means of air temperature . In a summer outdoor space, however, strong solar radiation from the sun gives a hot feeling, a strong wind gives a cool feeling, high humidity gives a muggy feeling, and a heated road surface feels so hot that it cannot be touched . Accordingly, it is necessary to include not only air temperature but also the environmental elements of thermal radiation, convection, humidity, and heat conduction in assessment of the thermal sense of humans . That is to say, it is necessary to make an explicit relationship between the thermal sense of the human body and the thermal environment evaluation indices that support the heat balance of the human body . Nikolopoulou and lykoudis, ishii et al ., and others show the relationship between the physical environmental factors of the outdoor space and the thermal sensation of a person and demonstrate that the outdoor environmental factors that influence the thermal sensation vote are air temperature, air velocity, and solar radiation . Clarify the effect that the environmental factors of a summer outdoor space have on sensation and physiological temperature . They demonstrate that the summer outdoor environmental factors that influence the thermal sensation of the human body are heat conduction, humidity, and short - wave solar radiation . They also demonstrate that the factors that affect the thermal comfort of the human body are air velocity, heat conduction, and humidity . In outdoor spaces, solar radiation from the sun and air velocity become strong, and the influence of sensational and physiological temperature increases . In addition, long - wave thermal radiation from the local objects on the ground surface forms a heterogeneous thermal radiation environment because of the effect of direct solar radiation, and the wind direction and current speed form a transient state that is constantly fluctuating . Accordingly, the effect on the human body of the physical environmental factors that compose sensational and physiological temperature is remarkably large in comparison with indoor space . Enhanced conduction - corrected modified effective temperature (etfe) and universal effective temperature (etu) are thermal environment evaluation indices that examine outdoor space and take solar radiation and heat conduction as environmental factors . Etfe and etu can temperature - convert the effect of air temperature and air velocity, long - wave thermal radiation in outdoor space, short - wave solar radiation, contact member's surface temperature, and humidity into individual meteorological elements . The addition of each temperature - such converted factor is also possible, and quantifying the composite effect on sensation in the outdoor space as well as the discrete effect of each meteorological element is possible on the same evaluation axis . Verification tests that demonstrate the relationship of the physiological and psychological effects on the human body have been carried out in summer for etfe, and its suitability as an outdoor environmental evaluation index has been demonstrated . Its correlation with outdoor thermal environmental factors in winter has not, however, been investigated . Accordingly, this research shows the relationship between the outdoor thermal environmental evaluation index for winter and the psychological response of the human body by using etfe to temperature - convert the effect of air temperature and air velocity, long - wave thermal radiation in outdoor space, short - wave solar radiation, contact member's surface temperature, and humidity and arrange them on the same evaluation axis . It goes on to investigate the individual environmental factors that should be incorporated as evaluation factors for an outdoor thermal environment . The thermal environment evaluation index for an outdoor space etfe was developed by kurazumi et al . . It was designed to include the influence of solar radiation and the concept of conduction - modified corrected operative temperature (etf). Etfe can temperature - convert the effect of air velocity and difference in attitude, long - wave thermal radiation in outdoor space, short - wave solar radiation, contact part's surface temperature, and humidity into individual meteorological elements . The effect of these five environmental factors on the heat balance of the human body can be expressed by a newly defined thermal environment evaluation index as follows: convective heat transfer area combined thermal velocity field for air velocity (tvfhta); radiant heat transfer area combined effective radiation field concerning long - wave thermal radiation in an outdoor space for long - wave thermal radiation in an outdoor space (erfhtal); radiant heat transfer area combined effective radiation field concerning short - wave solar radiation in an outdoor space for solar radiation (erfhtas); conductive heat transfer area combined effective conductive field for the contact member's surface temperature (ecfhta); and effective humid field in enhanced conduction - corrected modified effective temperature in humidity etfe for humidity (ehfetfe). The addition of each temperature - converted factor is also possible, and quantifying the composite effect on sensation in the outdoor space as well as the discrete effect of each meteorological element is possible on the same evaluation axis . Consider (1)etfe = ta+tvfhtahfl+erfhtalhfl+ecfhtahfl + ehfetfehfl+erfhtashfl,(2)tvfhta=(hofclfclofconvhcfcl fcl fconv)(tsta),(3)erfhtal = hrlfcl fcl frad(trlta),(4)ecfhta = hdfcld fcond(tfta),(5)ehfetfe = lwhcfcl fpcl(pa0.5petfe),(6)erfhtas = rs,(7)hfl = hofcl fclo fconv+hrlfcl fcl frad+hdfcld fcond, where etfe is the enhanced conduction - corrected modified effective temperature [k]; ta is the air temperature [k]; tvfhta is the convective heat transfer area of the combined thermal velocity field [w / m]; erfhtal is the radiant heat transfer area combined with the effective radiation field for long - wave radiation in outdoor space [w / m]; erfhtas is the radiant heat transfer area combined with the effective radiation field for short - wave solar radiation in outdoor space [w / m]; ecfhta is the heat transfer area combined effective conduction field [w / m]; ehfetfe is the effective humid field at enhanced conduction - corrected modified effective temperature [w / m]; hrl is the radiant heat transfer coefficient for long - wave radiation in outdoor space [w / mk]; fcl is the effective surface area factor of clothing []; fconv is the convective heat transfer area factor []; fcond is the conductive heat transfer area factor []; frad is the radiant heat transfer area factor []; fcl is the thermal efficiency factor of clothing in the exposed airflow area []; fcld is the thermal efficiency factor of clothing in the heat conduction area []; fclo is the thermal efficiency factor of clothing under standard conditions []; fpcl is the permeation efficiency factor of clothing []; hc is the convective heat transfer coefficient [w / mk]; hd is the resultant heat conductance [w / mk]; hfl is the sensible heat transfer coefficient in outdoor space [w / mk]; ho is the convective heat transfer coefficient under standard conditions [w / mk]; l: lewis relation coefficient [k / kpa]; pa is the water vapour pressure at outdoor air temperature [kpa]; petfe * is the saturated water vapour pressure at enhanced conduction - corrected modified effective temperature [kpa]; rs is the short - wave solar radiation heat gain in human body [w / m]; ts is the convection - corrected mean skin temperature [k]; tf is the surface temperature of the contacted material [k]; trl is the mean radiant temperature for long - wave radiation in outdoor space [k]; and w is the skin wetness []. Similarly to the measurement technique kurazumi et al . Used in summer, field observations were carried out on foot . As a trolley was used to transport the thermal environment measuring instruments, the movement speed was slower than walking speed at around 0.7 m / s . The observation points were drawn at random and the route to the points was not fixed . To reduce the burden on subjects, morning measurements were carried out from around 10:00 to 13:00, and afternoon measurements were carried out from around 13:00 to 16:00 . The thermal environment of winter outdoor spaces can be harsh to the extent that the body temperature drops to the zone of body cooling . Accordingly, one must avoid extended periods in outdoor spaces where strong winds prevail in a low - temperature environment . Consequently, because of the subjects' standing position and the response time of the assmann ventilated psychrometer, the actual measurement of the human body response and thermal environment in the mobile observations was performed after the observation device had been set up and left for five minutes . Naturally, it can be conjectured that the human body response will differ the longer the exposure time of the subjects, and the experimental period was determined with consideration for the safety of the subjects . Differently from indoor space, it is difficult to spend extended periods in an outdoor thermal environment as it would be uncomfortable because of behavioural thermoregulation by means of environmental refuge behaviour . Adaptation to the thermal environment according to the influence of thermal environment history is apparent, but a research method that removes the influence of environment history to the greatest possible extent was used in this research . Subjects moved on foot to the observation point after sitting and being at rest for 60 minutes or more in an indoor air - conditioned space at 22c room temperature and 40% humidity in order to suppress the environment history . While they were seated and at rest, the subjects were informed that psychological reporting involves a thermally specific sense of thermal sensation and thermal comfort, that they would be asked to report an average sense during the period of exposure, and that intake and excretion of liquids were prohibited until the conclusion of the experiment . The migration speed of the subjects was around 0.7 m / s as detailed above because of the movement of the trolley in which the research staff transported the measurement instruments . After arriving at each measurement point, the subjects waited in a standing posture for five minutes while the test staff set up the measurement instruments for the thermal environment and preparations for measurement were concluded . Thereafter, the subjects were exposed to the thermal environment in a standing posture for five minutes, as shown above . The subjects were positioned around 1.5 m away from the centre of the thermal environment measurement instruments in a location where they did not obstruct the sunlight and they surrounded the thermal environment measurement instruments . In the case of observation points 4 and 10, which were located on pavements, the subjects were positioned directly facing the road with the thermal environment measurement instruments as the focus because of space considerations . As the subject of the research was the environment surrounding the observation stations, the point of gaze of the subjects was free and unfixed . After five minutes' exposure, the subjects reported the average thermal sensation and the average thermal comfort for the whole body that they experienced while exposed at the observation point . 15 observation points were selected after consideration of the ground surface: bare ground where the surface was gravel or soil; paved ground such as concrete, asphalt, or blocks; green areas covered in plants; and water surfaces . The observation points were also selected with regard to the sky factor and the presence of buildings, trees, and so forth and the proportion of the solid angle of components of greenery, water, and so forth comprising the solid angle of the total celestial sphere (hereafter, green cover ratio). Their age was 22.0 2.1, their height was 157.5 3.4 cm, and their weight was 50.4 5.6 kg . With a bmi of 20.3 2.0, they can be considered to be unremarkable test subjects . In accordance with the helsinki declaration, the details of the experiment were explained to the subjects in advance, and their consent to participation in the experiment was obtained . In order to maintain consistency with the measurements kurazumi et al . Carried out in summer, air temperature and humidity, air velocity, short - wave solar radiation, long - wave thermal radiation, ground surface temperature, and water surface temperature were measured as thermal environment conditions . The air temperature and humidity were measured at a height of 0.9 m above the ground by means of an assmann ventilated psychrometer . The average air velocity was measured for five minutes at 1.2 m above the ground by a nondirectional hot - bulb air velocity sensor (kanomax japan, inc . : 6533, measurement range 0.05~30.0 m / s). Concerning the short - wave solar radiation in the regions from the visible to the near- and - mid - infrared and the long - wave thermal radiation of the terrestrial radiation in the far infrared region, radiation quantities in both the downwards and upwards directions were measured at a height of 0.9 m above the ground by long- and short - wave radiometers (eko instruments: mr-50, sensitivity 7 v / wm, short - wave range 305~2800 nm, long - wave range 5000~50000 nm). Ground surface temperature in the vicinity of the human body was measured by a radiation thermometer (konica minolta: ht-10d, measurement wave 8~14 m, measurement angle 1.4~2, emissivity measurement range 0.10~1.00). The sky factor was measured by a photograph of the sky taken 1.2 m above the ground at the observation point with a fisheye lens with an orthographical projection format (nikon: op fisheye nikkor 10 mm f/5.6) and a 35 mm digital slr camera . The proportion of the solid angle of components of greenery, water, and so forth comprising the solid angle of the total celestial sphere was measured by a photograph of the sky taken 1.2 m above the ground at the observation point with a fisheye lens with an equisolid angle projection format (olympus: fisheye zuiko 8 mm f/2.8) and a 35 mm digital slr camera . The albedo, sky temperature, and ground surface temperature were calculated from each directional component of the short - wave solar radiation and the long - wave thermal radiation . Furthermore, since the ground surface temperature in the vicinity of the human body is essential for the calculation of transmission heat quantity, values measured by radiation thermometer were used . Also, the values calculated by long- and short - wave solar radiometers were used as the average surface temperature and average sky temperature for the calculation of long - wave thermal radiation . With regard to the physiological conditions of the human body, the skin temperature of the part exposed to the air velocity was measured by a thermistor thermometer (nikkiso - therm, n542r and itp8391, measurement range 50~230c, resolution 0.01c), and the skin temperature of contact parts was measured by a heat flux temperature sensor (captec enterprise, hf series, 0.4 mm thick, sensitivity 1.692.10 mv/(w / m), response time 200 ms, t type thermocouple measurement range 50~230c, one side painted black). The temperature of skin exposed to the air velocity was measured at the seven positions of the head, trunk, arm, hand, thigh, lower leg, and foot . The sole of the foot was measured for the temperature of the contact part skin . The clothing quantity of the subjects was sought by the clo value by layering the clothing reported by the subjects . Psychological response was measured after subjects had stayed at the observation point for five minutes by rating the whole - body thermal sensation (cold - hot) and the whole - body thermal comfort (comfortable - uncomfortable) on a linear scale [29, 30]. Only a direction was given for each scale, and reported values were rated from zero to 100 . Etfe is an outdoor thermal environment evaluation index based on the heat balance of the human body . Accordingly, a weighting factor that takes into account the convection area of heat transfer surface was used for the calculation of the average skin temperature used to calculate the heat balance of the human body . Then, the average skin temperature used for the physiological response of the human body was calculated by means of a weighting factor that takes into account heat conduction . The values of kurazumi et al . Were used for the convective heat transfer area factor, the radiant heat transfer area factor and the conduction heat transfer area factor, for the human body . The value of miyamoto et al . Was used for the projection ratio of the human body . The values of kuwabara et al . Were used for the radiant heat transmissibility and convective heat transmissibility of the human body . 's value of 0.98 found for the reflectance of skin in electromagnetic waves of wavelength 3 m or more was used for the emissivity of the human body . 's value of 0.70 found for the reflectance of skin in electromagnetic waves of wavelength 3 m or less was used for the solar radiation absorption factor of the human body . The heat transfer of short - wave solar radiation is affected by the solar radiation absorption factor . According to vdi3787 - 2 watanabe et al . Showed, however, that the absorptivity of a human body wearing black clothes is 0.76 and that of one wearing white clothes is 0.38 . They also considered the solar radiation absorption factor for other combinations of clothing or ordinary clothes to be within the range of the absorptivity of a human body wearing black clothes and that of one wearing white clothes . In this research, the absorptivity of the human body was based on the naked body and taken to be 0.7 . With regard to skin wetness, values calculated by the two - node model were used because it was difficult to find the perspiration quantity . Etfe was calculated from weather observation values, the skin temperature of the human body, and clothing quantity . Although there is also an effect of air temperature, the effect of heating by short - wave solar radiation and cooling by radiation from the ground surface can be identified . Although the contact area between the human body in a standing position and the ground surface is small, the heat acquisition of the human body by heat conduction is conjectured to have a strong effect on the contact skin temperature . Air velocity was comparatively gentle at about 3 m / s throughout all observations . Accordingly, the influence that the difference in convection heat exchange has on sensational and physiological temperature is conjectured to be weak . In order to predict the thermal sensation votes for the human body, multiple linear regression analysis was performed with air temperature ta and air velocity, which are components of etfe tvfhta, long - wave thermal radiation erfhtal, heat conduction ecfhta, humidity ehfetfe, and short - wave solar radiation erfhtas as explanatory variables . The relationship between the measured thermal sensation votes and values predicted for the thermal sensation votes by the multiple regression equation is shown in figure 1 . Being negative, the partial regression constant for air velocity tvfhta had a different symbol from other environmental factors . The convection heat exchange because of air velocity tvfhta functions as a heat loss for the human body, but heat exchange owed to other environmental factors functions as a heat gain for the human body . Accordingly, air velocity controls the direction of increase or decrease of thermal sensation vote owed to other environmental factors, and so all environmental factors were employed as explanatory variables in the multiple regression formula . A laboratory experiment is an environment in which it is easy to control environmental stimuli and human body conditions . Office spaces permit a wider range of thermal environments than laboratory space controlled by a supervisor . In turn, the living space in which a broader range of behaviour is available to the individual permits a wider range of thermal environment than does the office space . Finally, outdoor space which may be thermally uncomfortable but in which one's selection of location or point of attention may be changed at will permits a broader range of thermal environment than living space . That is, there is a significant dispersion in the psychological response of the human body in outdoor space . The coefficient of determination for the multiple regression formula depends on the experimental judgement of the analyst . The coefficient of determination for the multiple regression formula found in this study was 0.48, and the multiple regression formula was considered to be quite good . Furthermore, p <0.01 as a result of analysis of variance on the multiple regression formula proved that the formula was valid . As a standard for selection of explanatory variables in order to derive multiple linear regression analysis results that are more useful in practice, the objective significance probability was sometimes made 2030% . Given that the psychological quantity for the human body in an outdoor environment differed from the case of an indoor environment and the results showed a lot of noise and dispersion, investigations were carried out with a significance probability of 25% . As a result of t - testing the components of etfe, it was found that the thermal sensation vote for outdoors in winter is strongly influenced by and varies according to air temperature ta, long - wave thermal radiation erfhtal, and short - wave solar radiation erfhtas . As stated in the previous results previous, the air velocity was comparatively gentle at about 3 m / s throughout all observations, and it is possible that the difference in convection heat exchange had little influence on sensational and physiological temperature . Also, in winter, footwear with thick soles and excellent heat insulation is generally worn in order to protect against the cold . Accordingly, it can be said that the heat acquisition of the human body because of heat conduction did not influence thermal sensation votes even though the ground surface temperature was low because of the effect of insulation . Dealing with the relationship between outdoor environmental elements and thermal sensation votes, givoni et al ., oliveira and andrade, eliasson et al ., nikolopoulou and steemers, nikolopoulou and lykoudis, and others considered that short - wave solar radiation, air velocity, and air temperature strongly influence thermal sensation votes . . Demonstrated that the summer outdoor environmental factors which influence the thermal sensation vote of the human body are heat conduction, humidity and short - wave solar radiation . Ishii et al ., who dealt with the relationship between outdoor environmental elements in winter and thermal sensation votes, considered air temperature, humidity, and short - wave solar radiation to have a strong influence on thermal sensation votes . In this research, the same trend is shown concerning air temperature and short - wave solar radiation; however, results different from those of ishii et al . Are shown for other factors . In order to predict the thermal comfort votes for the human body, multiple linear regression analysis was performed with air temperature ta and air velocity, which are components of etfe tvfhta, long - wave thermal radiation erfhtal, heat conduction ecfhta, humidity ehfetfe, and short - wave solar radiation erfhtas as explanatory variables . The results of the multiple linear regression analysis for the thermal comfort votes, which were the objective parameters, are shown in table 4 . The relationship between the measured thermal comfort votes and values predicted for the thermal comfort vote by the multiple regression equation is shown in figure 2 . Similarly to the relationship between the above components of etfe and thermal sensation votes, the partial regression constant for air velocity tvfhta differed from other environmental factors inasmuch that it was negative . Air velocity tvfhta controls the direction of increase or decrease of thermal comfort vote, however, because of other environmental factors, and so all environmental factors were employed as explanatory variables in the multiple regression formula . Although the analysis of the multiple regression formula cannot be said to be accurate, the multiple regression formula was shown to be valid, with p <0.01 resulting from analysis of variance on the multiple regression formula . The poor analytical accuracy may have been influenced by environmental factors other than thermal environment stimuli . In this research, the experiment was carried out after the subjects had been informed that comfort is specific to heat . It was, however, implied that there is a psychological tendency to perform a nonspecific and comprehensive evaluation of the entire space including visual and auditory stimuli, when one is outdoors [1014]. As a result of t - testing the components of etfe, it was shown that the thermal comfort vote for outdoors in winter is easily influenced by air temperature ta, humidity ehfetfe, short - wave solar radiation erfhtas, long - wave thermal radiation erfhtal, and heat conduction ecfhta . Considering that the air velocity was comparatively gentle at about 3 m / s throughout all observations, it is conjectured that the thermal comfort vote for outdoors in winter is influenced by all environmental factors addressed in this research . Accordingly, it may be necessary to treat short - wave solar radiation and heat conduction in the thermal environment evaluation of an outdoor space . Ishii et al . Demonstrated that the winter outdoor environmental factor which influences the thermal comfort vote of the human body is short - wave solar radiation . . Demonstrated that the summer outdoor environmental factors which influence the thermal comfort vote of the human body are air velocity, heat conduction, and humidity . Although it is physically possible to handle environmental stimuli, it is difficult to handle the human body effect on the environmental stimuli independently . For example, in the case of a hot environment in summer where the temperature is higher than skin temperature, the heat balance of the human body dropping as the air velocity increases can promote a sense of discomfort . In addition, in the case of a cooling winter environment of a temperature lower than the air temperature because of radiative cooling, the heat balance of the human body as the air velocity increases can show a thermal stimulus close to the air temperature . In the above, the response of the thermal sense to the environmental stimuli is subsidiary, but when the rate at which the environment stimuli contribute to thermal sense is investigated, it is appropriate to consider the following . In the evaluation of a winter outdoor environment, it has been demonstrated that it is essential to incorporate short - wave solar radiation and heat conduction as evaluation factors in addition to air temperature and air velocity, humidity, and long - wave thermal radiation . For air velocity measurement and winter outdoor environmental factors and those of kurazumi et al . For summer outdoor environmental factors, we have demonstrated that it is essential to include short - wave solar radiation and heat conduction in the evaluation of outdoor thermal environments . An experiment on test subjects was performed to clarify the effect that the thermal environment stimuli in a winter outdoor space have on the human body, and the relationship between outdoor environmental factors and the psychological response of the human body was demonstrated . In addition to air temperature and humidity, air velocity, and long - wave thermal radiation, it was found that short - wave solar radiation and heat conduction are among the influential factors which affect the thermal sensation vote and thermal comfort vote of the human body in a winter outdoor thermal environment . The validity of using a thermal environment evaluation index that incorporates these environmental factors for an outdoor space was demonstrated.
Two - component systems, which are found in large numbers within bacteria, offer a rich setting for exploring the evolution of cell signaling (wuichet et al, 2010). These systems are composed of a sensor kinase (sk) that receives input signals and controls the phosphorylation state of a partner response regulator (rr). Two - component systems generally show a high level of specificity, with sks controlling only their partner rrs (laub and goulian, 2007). Weak phosphorylation between non - partner proteins can be detected in vitro (skerker et al, 2005; yamamoto et al, 2005) but, in at least several examples, has been shown to be suppressed in vivo in wild - type strains (silva et al, 1998; siryaporn and goulian, 2008; groban et al, 2009). The weak interactions underlying this phosphorylation cross - talk may provide an efficient starting point for the evolution of new signaling circuits . However, signal transduction in many two - component systems depends on the sk mediating two distinct and antagonistic reactions: rr phosphorylation and dephosphorylation . The critical role played by these opposing reactions in controlling the level of rr phosphorylation could significantly increase the barrier to evolving a functional signaling system . In several systems, weak cross - phosphorylation between a non - partner sk and rr can be detected in vivo in strains for which specific partner regulators have been deleted (silva et al, 1998; siryaporn and goulian, 2008; groban et al, 2009). However, the output is blind to input stimulus to the sk (silva et al, 1998; siryaporn and goulian, 2008). Thus, cross - talk in these cases appears as a fixed offset in rr activity rather than weak signal transduction activity . Analysis of interacting partners have identified residues in sks and rrs that are important for controlling the specificity of interaction (white et al, 2007; burger and van nimwegen, 2008; skerker et al, 2008; weigt et al, 2009; bell et al, 2010; szurmant and hoch, 2010). By modifying specificity - determining residues, sk and rr variants that specifically interact with non - partner proteins have been engineered, effectively rewiring the flow of phosphorylation (skerker et al, 2008; bell et al, 2010). However, for bifunctional sks, re - engineering the specificity for phosphotransfer alone (as in skerker et al, 2008), without a comparable switch in phosphatase specificity, may not necessarily produce a new system that is signal responsive . Indeed, while engineered sk variants tested in vivo showed strong and specific phosphorylation of new partners (skerker et al, 2008), we have found that, at least for the cases that we tested, the variants were unresponsive to changes in input stimulus (supplementary figure s1). Taken together, the above observations suggest that to produce a signal - responsive system, it may be necessary to change both phosphotransfer and phosphatase specificity of a bifunctional sk . Here, we use a directed evolution approach to explore interactions between the non - partner regulators cpxa and ompr . We show that by combining mutations in cpxa that individually increase ompr phosphorylation, phosphatase activity emerges and results in a circuit that is responsive to changes in cpxa stimulus . To explore the connections between specificity and signal response, we screened for increased cross - talk between a pair of non - partner regulators in escherichia coli: cpxa and ompr (figure 1a). Cross - talk from cpxa to ompr is detectable in vitro (skerker et al, 2005) and in vivo in strains that lack the partners for these regulators, envz and cpxr (siryaporn and goulian, 2008). To identify amino - acid substitutions in cpxa that produce increased cross - talk, individual sites along the dhp domain of cpxa, the portion of the protein associated with sk rr interaction (zapf et al, 2000; marina et al, 2005; laub and goulian, 2007; casino et al, 2009; szurmant and hoch, 2010), were mutated by saturation mutagenesis . For each site, a library with the corresponding codon randomized was screened for increased expression of cfp from an ompr - regulated promoter and was characterized for its propensity to produce increased cross - talk (supplementary figure s2). Individual mutants that showed significantly increased cross - talk were selected from the libraries for five sites (i v supplementary figure s2b d) and analyzed in greater detail (figure 1). For all of these single - site mutants, the level of ompr - regulated transcription was comparable to or surpassed the level set by ompr's partner kinase envz (figure 1b). In addition, the effect of these mutants on transcriptional regulation by cpxr, the partner rr to cpxa, suggests the increased cross - talk is not due to a general hyperactivity of the mutants (supplementary figure s4). To provide further evidence that these mutants produce increased cross - talk to ompr, we used a previously developed assay for co - localization of an ompr - yfp protein fusion with plasmids containing ompr - binding sites in live cells (batchelor and goulian, 2006; siryaporn and goulian, 2008). We tested two mutants (substitutions at sites i and iii) using this assay . Both showed dramatically increased localization of ompr - yfp when compared with wild - type cpxa (figure 1c). An in vitro phosphorylation assay also demonstrated that these mutants phosphorylate ompr with significantly faster kinetics relative to wild - type cpxa (figure 1d; supplementary figure s5), again consistent with increased cross - talk to ompr . In an attempt to further increase cross - talk from cpxa to ompr, individual substitutions at sites i v were combined, resulting in cpxamut1 (figure 2a). This could indicate a defect in cpxa resulting from combining mutations or that the protein functions as a phosphatase . To test for evidence of phosphatase activity, we used a strain with an envz allele that encodes a kinase+ phosphatase envz mutant . Expression of wild - type cpxa (in the absence of cpxr) resulted in an increase in reporter gene expression, suggesting a further increase in ompr phosphorylation due to cross - talk from cpxa (figure 2b). We note that the kinase+ phosphatase envz mutant does not show the cross - talk suppression observed for wild - type envz (siryaporn and goulian, 2008; groban et al, 2009). This is consistent with a mechanism of cross - talk suppression that depends on the cycle of phosphorylation and dephosphorylation of the wild - type protein (siryaporn and goulian, 2008; groban et al, 2009). In contrast with the behavior of wild - type cpxa, expression of cpxamut1 resulted in a considerable decrease in ompr - regulated transcription (figure 2b). Similar results were observed in an envz strain in growth conditions that produced high levels of the phosphodonor acetyl phosphate (supplementary figure s6). Cpxa responds to envelope stress (raivio and silhavy, 2000) and is relatively unstimulated under ordinary growth conditions . Over - expression of the lipoprotein nlpe, a condition that stimulates cpxa (snyder et al, 1995), produced a small increase in ompr - regulated transcription in the cpxamut1 strain (figure 2c), suggesting cross - talk from cpxamut1 to ompr is weakly signal responsive . To improve the signal response, we performed a genetic screen using cpxamut1 as a starting point for mutagenesis . By screening for colonies that showed increased reporter gene expression upon induction of nlpe, we identified cpxamut2, which differs from cpxamut1 by a single substitution at site iv (figure 2a). In contrast to cpxamut1, cross - talk from cpxamut2 to ompr shows a strong dependence on activating and repressing stimuli (figure 2c; supplementary figure s7). Cpxamut2 also shows behavior in vivo consistent with phosphatase activity toward ompr - p (figure 2b; supplementary figure s6) and shows significant phosphatase activity toward ompr - p in an in vitro assay (figure 2d). It is also possible that cpxamut2 decreases ompc - cfp transcription by sequestering ompr - p, although we note that we have not detected localization of ompr - gfp to the periphery in cells expressing cpxamut2 at wild - type levels (data not shown). The above results suggest that cpxamut2 responds to input signals and functions as a bifunctional sk, phosphorylating and dephosphorylating ompr . For a bifunctional sk, rr phosphorylation is expected to be robust or insensitive to changes in sk expression level (batchelor and goulian, 2003; shinar et al, 2007; shinar and feinberg, 2010). This robust behavior is evident for cpxamut2, in marked contrast with the behavior of wild - type cpxa (figure 2e), providing further support that cpxamut2 functions as a bifunctional sk . Combining five amino - acid substitutions in cpxa, each of which individually increases ompr phosphorylation, produces a signal - responsive mutant that phosphorylates and dephosphorylates ompr . Remarkably, in the context of a simple model of the cycle of phosphorylation and dephosphorylation mediated by a bifunctional sk (figure 3a; see supplementary information for details), this complex change in phenotype can be accounted for by the change in a single parameter: the strength of the interaction or affinity between cpxa and ompr . We used this model to ask how rr phosphorylation level depends on the sk rr interaction strength . The model predicts qualitatively different behaviors for the limits of weak and strong interaction, corresponding to low and high affinity between sk and rr, respectively . In the weak - interaction limit, . With increasing interaction strength, rr phosphorylation first increases and then decreases (figure 3a, middle and right panels), indicating the emergence of phosphatase activity . This counterintuitive result reflects the asymmetry between sk - mediated phosphorylation and dephosphorylation of the rr: the phosphorylation reaction requires that the sk first autophosphorylates, whereas the dephosphorylation reaction does not require any modification of the sk . The model predicts that bifunctionality and signal responsiveness emerge in the strong - interaction limit . Rr interaction provides a simple interpretation of the cpxa mutants described above (figure 3a, right panel). In particular, the model suggests the emergence of phosphatase activity, signal responsiveness, and robustness in cpxamut2 can result simply from a greatly increased interaction with ompr . The model suggests that ompr interacts significantly more strongly with cpxamut2 than with wild - type cpxa . To test this, we over - expressed the sks in a strain expressing a fusion of gfp to the c - terminus of the ompr receiver domain (ompr'-gfp), the region that interacts with envz . Over - expression of envz produces increased fluorescence at the cell periphery (figure 3b), consistent with a strong interaction of the ompr receiver domain with the membrane protein envz . Over - expression of wild - type cpxa or a single - site mutant, on the other hand, showed a uniform distribution of fluorescence, which is consistent with a much weaker interaction of the ompr receiver domain with these membrane proteins . In particular, although the cpxa single - site mutant shows cross - talk to ompr that is comparable to that of envz (figure 1b), it nevertheless interacts relatively weakly with ompr . In contrast, over - expression of cpxamut2 produced strong fluorescence at the cell periphery, similar to the fluorescence distribution observed for over - expression of envz, suggesting a strong interaction with ompr and consistent with the predictions of the model (figure 3a). Over - expression of cpxamut1 produced fluorescence localization that was similar to that of cpxamut2 (data not shown). In the context of our model, the weaker signal response of cpxamut1 compared with cpxamut2 could be due to a stronger sk rr interaction for cpxamut1, although this effect is not distinguishable through our measurements of ompr'-gfp localization . The subtle synergistic and antagonistic interactions between amino acids in proteins present challenges for interpreting the relations between sequence, structure, and function in protein evolution and design (depristo et al, 2005; camps et al, 2007; bloom and arnold, 2009). Indeed, it is not uncommon to find that the outcome of combining multiple mutations in a gene cannot be easily predicted from the effects of each mutation alone . However, if a protein carries out more than one function, such as the phosphorylation and dephosphorylation reactions controlled by bifunctional sks, its activity may have a complex dependence on relatively simple physicochemical properties . This can give the appearance of complex intragenic interactions between mutations and produce dramatic changes in activity with relatively few amino - acid substitutions . Considering that phosphorylation cross - talk from cpxa to ompr is not detectable in wild - type cells (siryaporn and goulian, 2008), a remarkably small number of amino - acid substitutions were required to produce a robust signaling pathway connecting these two proteins . This is consistent with the above analysis, which suggests that the complex behavior resulting from combining mutations can be explained by a simple increase in the strength of interaction between sk and rr . Interestingly, weak cross - phosphorylation has been identified in vitro between numerous pairs of two - component systems (skerker et al, 2005; yamamoto et al, 2005). The suppression of cross - talk in vivo (silva et al, 1998; siryaporn and goulian, 2008; groban et al, 2009) presumably prevents these interactions from having a deleterious effect on fitness, enabling them to persist . Based on the above results, we suggest that this latent cross - talk may endow networks of bifunctional sks and rrs with a considerable level of evolutionary plasticity . When additional bifunctional sks or rrs appear by gene duplication or lateral gene transfer, new pathways could initially be established from basal cross - talk as signal - blind connections with fixed levels of phosphorylation . Strengthening the interactions leads to phosphatase activity, signal responsiveness, and insulation from competing phosphorylation sources (alves and savageau, 2003; siryaporn and goulian, 2008; groban et al, 2009). By analogy with our observations for cpxa and from our modeling work, this complex behavior may emerge by simply combining mutations that increase cross - phosphorylation, which would be a relatively simple path in protein sequence space for the evolution of new signaling systems . This also simplifies the process of screening for new signaling pathways, which may be useful for synthetic applications involving the engineering of new microbial signaling circuits . To explore the connections between specificity and signal response, we screened for increased cross - talk between a pair of non - partner regulators in escherichia coli: cpxa and ompr (figure 1a). Cross - talk from cpxa to ompr is detectable in vitro (skerker et al, 2005) and in vivo in strains that lack the partners for these regulators, envz and cpxr (siryaporn and goulian, 2008). To identify amino - acid substitutions in cpxa that produce increased cross - talk, individual sites along the dhp domain of cpxa, the portion of the protein associated with sk rr interaction (zapf et al, 2000; marina et al, 2005; laub and goulian, 2007; casino et al, 2009; szurmant and hoch, 2010), were mutated by saturation mutagenesis . For each site, a library with the corresponding codon randomized was screened for increased expression of cfp from an ompr - regulated promoter and was characterized for its propensity to produce increased cross - talk (supplementary figure s2). Individual mutants that showed significantly increased cross - talk were selected from the libraries for five sites (i v supplementary figure s2b d) and analyzed in greater detail (figure 1). For all of these single - site mutants, the level of ompr - regulated transcription was comparable to or surpassed the level set by ompr's partner kinase envz (figure 1b). In addition, the effect of these mutants on transcriptional regulation by cpxr, the partner rr to cpxa, suggests the increased cross - talk is not due to a general hyperactivity of the mutants (supplementary figure s4). To provide further evidence that these mutants produce increased cross - talk to ompr, we used a previously developed assay for co - localization of an ompr - yfp protein fusion with plasmids containing ompr - binding sites in live cells (batchelor and goulian, 2006; siryaporn and goulian, 2008). We tested two mutants (substitutions at sites i and iii) using this assay . Both showed dramatically increased localization of ompr - yfp when compared with wild - type cpxa (figure 1c). An in vitro phosphorylation assay also demonstrated that these mutants phosphorylate ompr with significantly faster kinetics relative to wild - type cpxa (figure 1d; supplementary figure s5), again consistent with increased cross - talk to ompr . In an attempt to further increase cross - talk from cpxa to ompr, individual substitutions at sites i v were combined, resulting in cpxamut1 (figure 2a). This could indicate a defect in cpxa resulting from combining mutations or that the protein functions as a phosphatase . To test for evidence of phosphatase activity, we used a strain with an envz allele that encodes a kinase+ phosphatase envz mutant . Expression of wild - type cpxa (in the absence of cpxr) resulted in an increase in reporter gene expression, suggesting a further increase in ompr phosphorylation due to cross - talk from cpxa (figure 2b). We note that the kinase+ phosphatase envz mutant does not show the cross - talk suppression observed for wild - type envz (siryaporn and goulian, 2008; groban et al, 2009). This is consistent with a mechanism of cross - talk suppression that depends on the cycle of phosphorylation and dephosphorylation of the wild - type protein (siryaporn and goulian, 2008; groban et al, 2009). In contrast with the behavior of wild - type cpxa, expression of cpxamut1 resulted in a considerable decrease in ompr - regulated transcription (figure 2b). Similar results were observed in an envz strain in growth conditions that produced high levels of the phosphodonor acetyl phosphate (supplementary figure s6). Cpxa responds to envelope stress (raivio and silhavy, 2000) and is relatively unstimulated under ordinary growth conditions . Over - expression of the lipoprotein nlpe, a condition that stimulates cpxa (snyder et al, 1995), produced a small increase in ompr - regulated transcription in the cpxamut1 strain (figure 2c), suggesting cross - talk from cpxamut1 to ompr is weakly signal responsive . To improve the signal response, we performed a genetic screen using cpxamut1 as a starting point for mutagenesis . By screening for colonies that showed increased reporter gene expression upon induction of nlpe, we identified cpxamut2, which differs from cpxamut1 by a single substitution at site iv (figure 2a). In contrast to cpxamut1, cross - talk from cpxamut2 to ompr shows a strong dependence on activating and repressing stimuli (figure 2c; supplementary figure s7). Cpxamut2 also shows behavior in vivo consistent with phosphatase activity toward ompr - p (figure 2b; supplementary figure s6) and shows significant phosphatase activity toward ompr - p in an in vitro assay (figure 2d). It is also possible that cpxamut2 decreases ompc - cfp transcription by sequestering ompr - p, although we note that we have not detected localization of ompr - gfp to the periphery in cells expressing cpxamut2 at wild - type levels (data not shown). The above results suggest that cpxamut2 responds to input signals and functions as a bifunctional sk, phosphorylating and dephosphorylating ompr . For a bifunctional sk, rr phosphorylation is expected to be robust or insensitive to changes in sk expression level (batchelor and goulian, 2003; shinar et al, 2007; shinar and feinberg, 2010). This robust behavior is evident for cpxamut2, in marked contrast with the behavior of wild - type cpxa (figure 2e), providing further support that cpxamut2 functions as a bifunctional sk . Combining five amino - acid substitutions in cpxa, each of which individually increases ompr phosphorylation, produces a signal - responsive mutant that phosphorylates and dephosphorylates ompr . Remarkably, in the context of a simple model of the cycle of phosphorylation and dephosphorylation mediated by a bifunctional sk (figure 3a; see supplementary information for details), this complex change in phenotype can be accounted for by the change in a single parameter: the strength of the interaction or affinity between cpxa and ompr . We used this model to ask how rr phosphorylation level depends on the sk rr interaction strength . The model predicts qualitatively different behaviors for the limits of weak and strong interaction, corresponding to low and high affinity between sk and rr, respectively . In the weak - interaction limit, . With increasing interaction strength, rr phosphorylation first increases and then decreases (figure 3a, middle and right panels), indicating the emergence of phosphatase activity . This counterintuitive result reflects the asymmetry between sk - mediated phosphorylation and dephosphorylation of the rr: the phosphorylation reaction requires that the sk first autophosphorylates, whereas the dephosphorylation reaction does not require any modification of the sk . The model predicts that bifunctionality and signal responsiveness emerge in the strong - interaction limit . Rr interaction provides a simple interpretation of the cpxa mutants described above (figure 3a, right panel). In particular, the model suggests the emergence of phosphatase activity, signal responsiveness, and robustness in cpxamut2 can result simply from a greatly increased interaction with ompr . The model suggests that ompr interacts significantly more strongly with cpxamut2 than with wild - type cpxa . To test this, we over - expressed the sks in a strain expressing a fusion of gfp to the c - terminus of the ompr receiver domain (ompr'-gfp), the region that interacts with envz . Over - expression of envz produces increased fluorescence at the cell periphery (figure 3b), consistent with a strong interaction of the ompr receiver domain with the membrane protein envz . Over - expression of wild - type cpxa or a single - site mutant, on the other hand, showed a uniform distribution of fluorescence, which is consistent with a much weaker interaction of the ompr receiver domain with these membrane proteins . In particular, although the cpxa single - site mutant shows cross - talk to ompr that is comparable to that of envz (figure 1b), it nevertheless interacts relatively weakly with ompr . In contrast, over - expression of cpxamut2 produced strong fluorescence at the cell periphery, similar to the fluorescence distribution observed for over - expression of envz, suggesting a strong interaction with ompr and consistent with the predictions of the model (figure 3a). Over - expression of cpxamut1 produced fluorescence localization that was similar to that of cpxamut2 (data not shown). In the context of our model, the weaker signal response of cpxamut1 compared with cpxamut2 could be due to a stronger sk rr interaction for cpxamut1, although this effect is not distinguishable through our measurements of ompr'-gfp localization . The subtle synergistic and antagonistic interactions between amino acids in proteins present challenges for interpreting the relations between sequence, structure, and function in protein evolution and design (depristo et al, 2005; camps et al, 2007; bloom and arnold, 2009). Indeed, it is not uncommon to find that the outcome of combining multiple mutations in a gene cannot be easily predicted from the effects of each mutation alone . However, if a protein carries out more than one function, such as the phosphorylation and dephosphorylation reactions controlled by bifunctional sks, its activity may have a complex dependence on relatively simple physicochemical properties . This can give the appearance of complex intragenic interactions between mutations and produce dramatic changes in activity with relatively few amino - acid substitutions . Considering that phosphorylation cross - talk from cpxa to ompr is not detectable in wild - type cells (siryaporn and goulian, 2008), a remarkably small number of amino - acid substitutions were required to produce a robust signaling pathway connecting these two proteins . This is consistent with the above analysis, which suggests that the complex behavior resulting from combining mutations can be explained by a simple increase in the strength of interaction between sk and rr . Interestingly, weak cross - phosphorylation has been identified in vitro between numerous pairs of two - component systems (skerker et al, 2005; yamamoto et al, 2005). The suppression of cross - talk in vivo (silva et al, 1998; siryaporn and goulian, 2008; groban et al, 2009) presumably prevents these interactions from having a deleterious effect on fitness, enabling them to persist . Based on the above results, we suggest that this latent cross - talk may endow networks of bifunctional sks and rrs with a considerable level of evolutionary plasticity . When additional bifunctional sks or rrs appear by gene duplication or lateral gene transfer, new pathways could initially be established from basal cross - talk as signal - blind connections with fixed levels of phosphorylation . Strengthening the interactions leads to phosphatase activity, signal responsiveness, and insulation from competing phosphorylation sources (alves and savageau, 2003; siryaporn and goulian, 2008; groban et al, 2009). By analogy with our observations for cpxa and from our modeling work, this complex behavior may emerge by simply combining mutations that increase cross - phosphorylation, which would be a relatively simple path in protein sequence space for the evolution of new signaling systems . This also simplifies the process of screening for new signaling pathways, which may be useful for synthetic applications involving the engineering of new microbial signaling circuits . A detailed description of the experimental procedures, with specific references to each figure, is given in supplementary information.
Dietary sugar intake, in the form of sucrose or high - fructose corn syrup (hfcs), has dramatically increased and correlates with a rise in obesity, metabolic syndrome, and diabetes . Because a broad range of physiological, behavioural, and neurological variables influences food choices and eating behaviour, it is difficult to understand the mechanisms of eating behaviour and their alterations . The hedonic value of highly palatable foods and their wide availability can override the physiological mechanisms related to energy homeostasis [2, 3]. The hedonic reward value of food is closely linked to the sensory perception of food (including food taste, odour, and texture) and refers to the driving force behind the motivation to eat . The nutrient detection by the gut is mainly controlled by enteroendocrine (ee) cells and might activate a cascade of physiological phenomena, including endocrine regulations (e.g. Insulin, leptin, glucagon - like peptide-1 or glp-1, secretion), inhibition of gastric emptying, inhibition of food intake, stimulation of intake as well as psychobehavioural responses . Dietary sugar overconsumption might provoke deleterious effects at both central and peripheral levels, including alterations in (i) the regulation of secretion of satiety peptides and neuropeptides [7, 8]; (ii) gut permeability leading to low - grade inflammation and liver disease; (iii) blood brain barrier (bbb) permeability; (iv) the endocannabinoid, opioid, and mesolimbic dopaminergic systems, as well as (v) brain structures involved in reward processing . Both drugs and food have powerful reinforcing effects partly mediated by dopamine increases in the limbic system that, under certain circumstances or in vulnerable individuals, could overwhelm the brain s homeostatic control mechanisms, but the plausibility of sugar addiction and its role in obesity and eating disorders in humans is still a subject of controversy . Much of the research on the effects of dietary sugars on health has recently focused on fructose, given the striking parallel increases in obesity and in fructose intake over the past decades . These studies have found important fructose - induced health disturbances that are different from those provoked by glucose or sucrose . Most of fructose intake in diets originates from sucrose (containing 50% fructose and 50% glucose) and soft drinks containing high - fructose corn syrup (hfcs) (range 4765% fructose, and 5335% glucose). An estimate of the consumption of hfcs from beverages indicates a daily range between 132 and 316 kcal for americans aged over 2 years, and patients with non - alcoholic fatty liver disease (nafld) consume twofold more calories from hfcs from beverages than healthy patients (365 vs. 170 kcal / day). In the united states, average fructose consumption from sugar - sweetened beverages has increased from 37 to 49 g / day during the last 30 years (+ 0.4% per year). Thus, it is not clear whether the fructose - induced metabolic disturbances observed in human and animal studies are due to fructose itself or the associated increase in energy intake . Moreover, since fructose and glucose intake may vary simultaneously, this raises the consideration that other dietary sugars (e.g. Sucrose and glucose) might also contribute to the development of obesity and associated co - morbidities . In fact, there are controversial findings on metabolic effects between the different sugars (i.e. Glucose, fructose, and sucrose). While some studies have disclosed significant differences between these sugars, other studies have found small or no difference . For example, in overweight or obese humans, intake of a fructose - sweetened beverage led to a significant increase in visceral adipose tissue, hepatic de novo lipogenesis, and postprandial triglycerides compared to subjects offered a glucose - sweetened beverage . In lean and obese subjects, de novo lipogenesis increased to the same extent after overfeeding with glucose and sucrose . Both high - glucose and high - fructose diets stimulated lipogenic gene expression in rodents . Most of the studies on the effects of sugars on health and disease, at both peripheral and central levels, have been performed in rodents, and studies are missing in humans or other animal models closer to humans, such as the pig model . Given that human studies are limited due to ethical considerations, future studies should privilege the use of animal models that closely resembles humans . On the other hand, there is an impressive number of studies available concerning the effects of dietary sugars, using different experimental paradigms, with different approaches, animal models, oral intake doses, in the form of sugar solutions or added in the diet, or peripheral or central administrations . Therefore, it becomes extremely difficult to interpret and find a definite conclusion on these effects . In this context, we considered essential to gather all the information available to give a global view of the current research in this domain, in order to highlight the need to reformulate the questions and approach to these questions, under similar conditions between studies, and using integrated approaches, from the molecular to the behavioural level . The main goal of this review was to provide an overview of the impact of different dietary sugars on peripheral and central functions . It will gather (i) results from studies regarding the effects of sucrose, glucose, and/or fructose on metabolism, eating behaviour, and brain responses; (ii) current available data comparing the effects of these sugars, at both peripheral and central levels . It will also propose some clues and hypotheses for future research perspectives regarding the effects of these sugars, with special focus on fructose and glucose . Even though this review synthetises data from different animal models and humans, we wanted to dedicate a short section to the presentation of the pig model, which is of particular interest in nutrition and neurosciences . Pigs have emerged as an ideal model for nutritional and biomedical research because of their anatomical and physiological similarities to humans [2022], as well as blood chemical and biochemical characteristics, plasma hormone levels, and energy metabolism . Pigs are able to distinguish the palatability of different diets, and they have a high innate preference for sweet taste and a strong appetite for sugar solutions . The digestive system of pigs has anatomical differences with that of humans; however, the physiology of digestion is essentially similar . The pig has been used extensively as a model of digestion in connection with nutrition (and determination of food value) of the pig and for studying human digestive phenomena . The metabolic functions, intestinal transit times, and characteristics of nutrients absorption have made them useful in basic nutritional research . Other specific functional characteristics of swine that relate directly to humans include ion transport and motility, neonatal development of the gastrointestinal tract, and splanchnic blood flow characteristics . Like humans, these physiological characteristics of the gastrointestinal tract are probably due to the omnivorous diet they consume, unlike that of carnivores, ruminants, rabbits, and rodents . Similar to carbohydrate (sucrose and starch) digestion in humans, it was shown in non - anaesthetised mature pigs following the intake of different sugar - containing meals (with glucose, sucrose, lactose, or maize starch) that the absorption pattern was different for each sugar . The kinetics of appearance of glucose and of sucrose hydrolysis products in the portal blood were faster for glucose and sucrose than for sugars resulting from maize starch hydrolysis . Recent studies have shown the convenience for the use of pig model in brain imaging, behavioural, and physiological effects of obesity induced by highly palatable diets [20, 22, 28, 29]. Compared to the rodent brain, the pig brain more closely resembles the human brain in terms of anatomy and biochemistry, which associates the pig with a higher translational value . Several brain disorders have been fully or partially modelled in the pig, and this has further spurred an interest in having access to behavioural tasks for pigs and in particular to cognitive tasks . Cognitive testing of pigs has been conducted for several years in animal science, but it has only recently received interest in the wider neuroscience community . Several behavioural tasks have successfully been adapted to the pig, and valuable results have been produced . Aside of having similar brain structures to humans, the pig might develop metabolic disorders observed in humans (excessive fat deposition, diabetes, atherosclerosis, hypertension). Taken together, these data position the pig model as a valuable model for biomedical studies in nutrition and neuroscience . Therefore, future studies on the effects of dietary sugars on health and disease should favour the use of the pig model in order to extrapolate data to humans and propose modifications in the nutritional recommendations for humans . Gut microbiota operates like a metabolic organ, influencing nutrient availability and uptake, energy homeostasis, and the control of body weight . Diet composition may strongly influence changes in the microbiota, which in turn, when subjected to deleterious nutritional environment, might affect intestinal permeability and result in low - grade inflammation, obesity, and associated chronic metabolic diseases such as nafld, dyslipidaemia, and insulin resistance [9, 32]. Increases in gut permeability, low - grade endotoxemia (provoked by increased plasma lipopolysaccharides lps), and hepatic lipid accumulation have been reported in animal models of obesity induced by high - fat or high - fructose diets . A high - fructose diet has been associated with hepatic and extra - hepatic insulin resistance and obesity - related metabolic disturbances through a mechanism implicating gut microbiota and its effects on intestinal permeability . The hepatic manifestation of the metabolic syndrome is nafld, starting from simple steatosis and ending as liver cirrhosis . Dietary sugar intake might participate to nafld pathogenic history, and the type of sugar (e.g. Fructose) may affect the development of the disease . Consistent evidence has demonstrated the implication of fructose or hfcs in the development of nafld in several animal models [17, 3338]. The effects of dietary sugars, especially fructose, on the development of hepatic steatosis, liver damage, and other features of the metabolic syndrome found in several animal models are presented in table 1.table 1effects of dietary sugars intake on hepatic steatosis, liver damage, and various features of the metabolic syndrome in several animal models and in humansdietary interventiondurationhepatic steatosis / liver injuryplasma or hepatic measurementsother effectsmodelanimal studies20% hfcs in maternal diet20% hfcs + msg9 monthshepatic steatosis hepatic and serum tg, serum ffa srebf2 expressionc57bl/6 j micecollison high - fat / high - fructose diet:(a) 8% trans - fat, 20% hfcs (b) trans - fat (8%), hfcs (20%), 1% msg9 months alp, alt serum creatinine, cortisol serum leptin and fasting insulindomestic cat (male, n = 18)collison (a) trans - fat (9%) + hfcs (24%) (b) trans - fat (9%), hfcs (24%), 1% msg9 monthsfibrous expansion markers of hepatic fibrosis, angiogenesis, hepatocellular carcinoma alp and altcreatinine and cortisol (a)domestic cat (male, n = 18)collison 30% glucose solution30% fructose solution8 weekslipid accumulation endotoxin levels in portal bloodtnf higher with fructose than glucoseantibiotics prevented some effects of fructosec57bl / j6 micebergheim fructose + antibiotics 30% fructose solution (f)tap water (c)30% fructose solution + antibiotics polymyxin b and neomycin (ab)8 weekshepatic fat accumulation and tnf and inos mrna expression in liver with f suppressed by ab endotoxin tlr4 receptors, suppressed by ab tight junction occludin in duodenum (f)c57bl / j6 micewagnerberger 30% fructose solution8 weeksinduction of hepatic steatosis and inflammation alt levels hepatic lipidperoxidation, phospho-, nf- b and tnfc57bl / j6 micespruss fructose group: 10.5% fat 20% fructoseatherogenic diet: 20% fructose 46% fat, 2% cholesterol, 0.7% choline24 weeksfructose diet: normal liver histologyatherogenic diet: abnormal liver histology with microvesicular steatosis and fatty kupffer cells but not fibrosisfructose group: body weight, hypertension, and insulin resistanceatherogenic diet: metabolic syndromeosabaw minipiglee standard chow diet: (s)41% starch, 5% sugar, 4.5% fat, iron (120 mg / kg)fructose - enriched diet (f) 60% fructose, 5% fat + iron (5 mg / kg)5 weeks f: higher liver lipids, tg, and cholesterol than s group mild - to - moderate deposition of macrovesicular and microvesicular fat similar hepatic iron concentration in both groupsf: tg, insulin, homa scoref: blood pressuresprague dawley rats n = 49ackerman dietary interventiondurationhepatic steatosis / liver injuryplasma or hepatic measurementsother effectsrecruitment criteriahuman studiesusual ad libitum diet + fructose (n = 16) or glucose (n = 15) sweetened beverages at 25% of energy requirements10 weeksnot investigatedfructose group: fasting plasma mcp-1, pai-1, and e - selectin postprandial pai-1bmi: 2535 kg / m healthy,4072 yearcox usual ad libitum diet + fructose (n = 17) or glucose (n = 15) sweetened beverages at 25% of energy requirements10 weeks fractional hepatic de novo lipogenesis only in fructose groupfructose group: plasma lipid and lipoprotein postprandial tg, lipoproteins, and ldl cholesterolglucose group: fasting tg and ffa in glucose group postprandial lipoprotein lipase activity body weight, fat mass in both groups abdominal and visceral adipose tissue only in fructose group insulin sensitivity only in fructose groupbmi: 2535 kg / m healthy, 4072 yearstanhope hfcs high - fructose corn syrup, tg triglycerides, srebf2 sterol regulatory element - binding transcription factor 2, alp alkaline phosphatase, alt alanine aminotransferase, tnf tumour necrosis factor alpha, inos inducible nitric oxide synthase, ab antibiotics, nf- nuclear factor, tlr toll - like receptors, homa homeostatic model assessment, mcp-1 monocyte chemoattractant protein 1, pai-1 plasminogen activator inhibitor type 1, ffa free fatty acids effects of dietary sugars intake on hepatic steatosis, liver damage, and various features of the metabolic syndrome in several animal models and in humans hfcs high - fructose corn syrup, tg triglycerides, srebf2 sterol regulatory element - binding transcription factor 2, alp alkaline phosphatase, alt alanine aminotransferase, tnf tumour necrosis factor alpha, inos inducible nitric oxide synthase, ab antibiotics, nf- nuclear factor, tlr toll - like receptors, homa homeostatic model assessment, mcp-1 monocyte chemoattractant protein 1, pai-1 plasminogen activator inhibitor type 1, ffa free fatty acids it seems that hfcs or fructose exposure is able to induce hepatic steatosis, liver dysfunction, hepatic fibrosis as well as several features of the metabolic syndrome and inflammation in rodents and cats (e.g. [3944]). However, in other species such as pigs or in humans, this concept remains incompletely clear . Data obtained from humans and osabaw minipigs suggested that it is the association between high - fructose intake with other components in the diet, such as glucose, sucrose, fat, and cholesterol, responsible for the development of the metabolic syndrome and liver steatosis, rather than high - fructose intake itself . The approach used in several human studies where fructose daily intake pattern is assessed in patients with previously established hepatic steatosis [33, 46, 47] is not the best way to evaluate fructose as a risk factor for nafld . This question should be addressed in a more controlled experimental paradigm where dietary intake is closely monitored . To our knowledge, one of the few human studies that has assessed the effect of glucose- or fructose - sweetened beverages on the development of hepatic de novo lipogenesis under controlled conditions is the one performed by stanhope et al . . However, this study did not confirm the presence of hepatic steatosis using standard diagnostic methods such as mri, ct scan, or liver biopsy . Thus, studies in humans are needed to investigate the effects of dietary sugars on the development of hepatic steatosis under controlled experimental conditions . Taken together, studies with rodents and cats suggest that fructose induces liver damage, in part through mechanisms involving intestinal bacterial overgrowth, increased intestinal permeability, inflammation, and metabolic endotoxemia . However, underlying mechanisms explaining how fructose leads to bacterial overgrowth, inflammation, and increases in intestinal permeability remain poorly understood . Since it is difficult to achieve controlled experimental conditions in humans, for ethical reasons, studies in animal models closer to humans, e.g. Pigs [20, 28, 31], are a valuable approach that allows close monitoring of dietary interventions . If similar mechanisms occur in humans and pigs, novel strategies including low - fructose diets might be considered for the prevention / management of nafld . However, there seems to be substantial differences between rodents or cats and humans or pig studies . Thus, in the absence of clear evidence for a detrimental role for fructose, there is no justification for replacing it with other dietary sugars such as glucose or sucrose in human diets for the prevention of hepatic steatosis . The hepatic manifestation of the metabolic syndrome is nafld, starting from simple steatosis and ending as liver cirrhosis . Dietary sugar intake might participate to nafld pathogenic history, and the type of sugar (e.g. Fructose) may affect the development of the disease . Consistent evidence has demonstrated the implication of fructose or hfcs in the development of nafld in several animal models [17, 3338]. The effects of dietary sugars, especially fructose, on the development of hepatic steatosis, liver damage, and other features of the metabolic syndrome found in several animal models are presented in table 1.table 1effects of dietary sugars intake on hepatic steatosis, liver damage, and various features of the metabolic syndrome in several animal models and in humansdietary interventiondurationhepatic steatosis / liver injuryplasma or hepatic measurementsother effectsmodelanimal studies20% hfcs in maternal diet20% hfcs + msg9 monthshepatic steatosis hepatic and serum tg, serum ffa srebf2 expressionc57bl/6 j micecollison high - fat / high - fructose diet:(a) 8% trans - fat, 20% hfcs (b) trans - fat (8%), hfcs (20%), 1% msg9 months alp, alt serum creatinine, cortisol serum leptin and fasting insulindomestic cat (male, n = 18)collison (a) trans - fat (9%) + hfcs (24%) (b) trans - fat (9%), hfcs (24%), 1% msg9 monthsfibrous expansion markers of hepatic fibrosis, angiogenesis, hepatocellular carcinoma alp and altcreatinine and cortisol (a)domestic cat (male, n = 18)collison 30% glucose solution30% fructose solution8 weekslipid accumulation endotoxin levels in portal bloodtnf higher with fructose than glucoseantibiotics prevented some effects of fructosec57bl / j6 micebergheim fructose + antibiotics 30% fructose solution (f)tap water (c)30% fructose solution + antibiotics polymyxin b and neomycin (ab)8 weekshepatic fat accumulation and tnf and inos mrna expression in liver with f suppressed by ab endotoxin tlr4 receptors, suppressed by ab tight junction occludin in duodenum (f)c57bl / j6 micewagnerberger 30% fructose solution8 weeksinduction of hepatic steatosis and inflammation alt levels hepatic lipidperoxidation, phospho-, nf- b and tnfc57bl / j6 micespruss fructose group: 10.5% fat 20% fructoseatherogenic diet: 20% fructose 46% fat, 2% cholesterol, 0.7% choline24 weeksfructose diet: normal liver histologyatherogenic diet: abnormal liver histology with microvesicular steatosis and fatty kupffer cells but not fibrosisfructose group: body weight, hypertension, and insulin resistanceatherogenic diet: metabolic syndromeosabaw minipiglee standard chow diet: (s)41% starch, 5% sugar, 4.5% fat, iron (120 mg / kg)fructose - enriched diet (f) 60% fructose, 5% fat + iron (5 mg / kg)5 weeks f: higher liver lipids, tg, and cholesterol than s group mild - to - moderate deposition of macrovesicular and microvesicular fat similar hepatic iron concentration in both groupsf: tg, insulin, homa scoref: blood pressuresprague dawley rats n = 49ackerman dietary interventiondurationhepatic steatosis / liver injuryplasma or hepatic measurementsother effectsrecruitment criteriahuman studiesusual ad libitum diet + fructose (n = 16) or glucose (n = 15) sweetened beverages at 25% of energy requirements10 weeksnot investigatedfructose group: fasting plasma mcp-1, pai-1, and e - selectin postprandial pai-1bmi: 2535 kg / m healthy,4072 yearcox usual ad libitum diet + fructose (n = 17) or glucose (n = 15) sweetened beverages at 25% of energy requirements10 weeks fractional hepatic de novo lipogenesis only in fructose groupfructose group: plasma lipid and lipoprotein postprandial tg, lipoproteins, and ldl cholesterolglucose group: fasting tg and ffa in glucose group postprandial lipoprotein lipase activity body weight, fat mass in both groups abdominal and visceral adipose tissue only in fructose group insulin sensitivity only in fructose groupbmi: 2535 kg / m healthy, 4072 yearstanhope hfcs high - fructose corn syrup, tg triglycerides, srebf2 sterol regulatory element - binding transcription factor 2, alp alkaline phosphatase, alt alanine aminotransferase, tnf tumour necrosis factor alpha, inos inducible nitric oxide synthase, ab antibiotics, nf- nuclear factor, tlr toll - like receptors, homa homeostatic model assessment, mcp-1 monocyte chemoattractant protein 1, pai-1 plasminogen activator inhibitor type 1, ffa free fatty acids effects of dietary sugars intake on hepatic steatosis, liver damage, and various features of the metabolic syndrome in several animal models and in humans hfcs high - fructose corn syrup, tg triglycerides, srebf2 sterol regulatory element - binding transcription factor 2, alp alkaline phosphatase, alt alanine aminotransferase, tnf tumour necrosis factor alpha, inos inducible nitric oxide synthase, ab antibiotics, nf- nuclear factor, tlr toll - like receptors, homa homeostatic model assessment, mcp-1 monocyte chemoattractant protein 1, pai-1 plasminogen activator inhibitor type 1, ffa free fatty acids it seems that hfcs or fructose exposure is able to induce hepatic steatosis, liver dysfunction, hepatic fibrosis as well as several features of the metabolic syndrome and inflammation in rodents and cats (e.g. [3944]). However, in other species such as pigs or in humans, this concept remains incompletely clear . Data obtained from humans and osabaw minipigs suggested that it is the association between high - fructose intake with other components in the diet, such as glucose, sucrose, fat, and cholesterol, responsible for the development of the metabolic syndrome and liver steatosis, rather than high - fructose intake itself . The approach used in several human studies where fructose daily intake pattern is assessed in patients with previously established hepatic steatosis [33, 46, 47] is not the best way to evaluate fructose as a risk factor for nafld . This question should be addressed in a more controlled experimental paradigm where dietary intake is closely monitored . To our knowledge, one of the few human studies that has assessed the effect of glucose- or fructose - sweetened beverages on the development of hepatic de novo lipogenesis under controlled conditions is the one performed by stanhope et al . . However, this study did not confirm the presence of hepatic steatosis using standard diagnostic methods such as mri, ct scan, or liver biopsy . Thus, studies in humans are needed to investigate the effects of dietary sugars on the development of hepatic steatosis under controlled experimental conditions . Taken together, studies with rodents and cats suggest that fructose induces liver damage, in part through mechanisms involving intestinal bacterial overgrowth, increased intestinal permeability, inflammation, and metabolic endotoxemia . However, underlying mechanisms explaining how fructose leads to bacterial overgrowth, inflammation, and increases in intestinal permeability remain poorly understood . Since it is difficult to achieve controlled experimental conditions in humans, for ethical reasons, studies in animal models closer to humans, e.g. Pigs [20, 28, 31], are a valuable approach that allows close monitoring of dietary interventions . If similar mechanisms occur in humans and pigs, novel strategies including low - fructose diets might be considered for the prevention / management of nafld . However, there seems to be substantial differences between rodents or cats and humans or pig studies . Thus, in the absence of clear evidence for a detrimental role for fructose, there is no justification for replacing it with other dietary sugars such as glucose or sucrose in human diets for the prevention of hepatic steatosis . The regulation of food intake and energy homeostasis is achieved by a complex network communication between the periphery (e.g. Gut, liver, stomach, pancreas, and adipose tissue) and the brain . The different molecular structures of dietary sugars might result in different gastrointestinal peptide secretion profiles, leading to different metabolic and endocrine effects at both peripheral (e.g. Gut and liver) and central (e.g. Hypothalamus) levels [5254]. It has been shown that fructose, compared to glucose intake, produces smaller increases in plasma glucose and circulating satiety hormones, i.e. Insulin, leptin, glucagon - like peptide-1 (glp-1), peptide tyrosine tyrosine (pyy), and attenuates postprandial suppression of ghrelin [37, 5559]. This suggested an endocrine mechanism by which fructose might induce a positive energy balance and weight gain . A possible explanation of smaller increases in satiety hormones by fructose could be the lower expression of glut5 in -cells or lower absorption rates in the intestine . In addition, it was found that central administration of fructose provokes feeding in rodents, whereas centrally administered glucose promotes satiety [54, 62]. These data together with parallel increases between fructose intake and obesity development have led to the fructose hypothesis which postulates that fructose, compared to glucose, may stimulate food - seeking behaviour, food intake, and body weight gain . However, the proposed effect of fructose on the induction of feeding is the subject of debate since this concept has not been replicated in rodents, and there is little evidence linking these phenomena in humans [56, 58, 59, 63, 64]. The few studies linking fructose consumption with increased body weight compared fructose versus an artificial sweetener, evaluated 60-g fructose supplementation but did not compare it versus another sugar, or used higher doses of fructose compared sucrose . Even others found no substantial differences in endocrine and metabolic effects after consumption of sugar - sweetened beverages with hfcs, sucrose, fructose, and glucose in humans or in body weight and food intake or found greater increase in body adiposity with sucrose than with fructose solutions . Moreover, it appears that fructose orally ingested may cross the bbb to a small extent compared to glucose, which may have two opposite implications: 1) the limited fructose access to glucose - sensing neurons could contribute to the deregulation of food intake and energy balance, or 2) fructose might have no effect at all on appetite regulation due to the lack of fructose transport to the brain . The purpose of this section was to present available data on the main enteric and cephalic detection processes of dietary sugars, in association with satiety peptides, neuropeptides secretion, and neuronal activity, and to discuss their effects on food intake . Peripheral sweet taste and sugar detectors are key regulators of feeding behaviour and energy homeostasis . Taste - signalling mechanisms identified in the oral epithelium also operate in the gut and play a role in both sugar detection and regulation of intestinal and pancreatic hormone secretion . There are two main groups of sugar detectors: members of the g - protein - coupled receptors (gpcr) family, and sugar transporters (e.g. Glut2, glut5, sodium - dependent glucose co - transporter 1 (sglt1), and glut8). Enteroendocrine cells directly sense sugars via gpcr, including the sweet taste receptors of type 1, t1r . The initial step in taste recognition occurs on the apical surface of taste receptor cells, within taste buds of the tongue and palate . The regulation of taste sensitivity by appetite peptides at the level of taste bud cells in the tongue as well as in enteroendocrine cells of the taste epithelium may be important in the control of eating behaviour and the regulation of energy homeostasis . However, this concept remains unclear since it was found that knockout (ko) mice (p2x2/p2x3) with adenosine triphosphate taste cell signalling deficits show relatively normal food intake and body weight . They also develop strong preferences for non - taste nutrients by the post - oral actions of these nutrients . Furthermore, much of the research on appetite peptides and taste detection in the mouth has been performed at the cell / neuron level; however, little empirical evidence exists to date for these peptides impacting taste function at the level of the mouth . Therefore, more studies should be performed in large animal models to clarify these concepts . Appetite regulatory peptides, such as leptin, endocannabinoids, glp-1, glucagon, oxytocin, insulin, cholecystokinin (cck), neuropeptide y (npy), and vasoactive intestinal peptide (vip), modulate taste sensitivity at the level of oral sweet taste cells . Leptin selectively suppressed sweet taste responses of cells isolated from circumvallate papillae from non - diabetic mice, but not in diabetic db / db mice . This indicated that the effect of leptin on sweet taste sensitivity is mediated by the leptin receptor expressed in these cells . The t1r3 subunit is co - expressed with the leptin receptor in both fungiform and circumvallate taste bud cells, and leptin suppresses sweet taste sensitivity in mice by affecting responsiveness of t1r3-expressing taste cells via the leptin receptor . Therefore, leptin may play an important role in the regulation of sweet taste sensitivity in the tongue, besides its central actions on food intake . Co - expression of glp-1 with taste - signalling elements such as t1r2, t1r3, and -gustducin, a gi family member associated with taste perception, was found in human intestinal endocrine l cells . These taste - signalling elements mediate the glucose - dependent secretion of glp-1 and maintain or enhance sweet taste sensitivity via paracrine action . In addition to its intestinal expression, glp-1 was also found to be expressed in taste cells in mouse circumvallate papillae taste buds; it was co - expressed with -gustducin and t1r3-expressing sweet taste cells in mouse taste buds, and it was produced in taste buds from lingual extracts in its active form . In contrast to its presence in blood and ileum, dipeptidyl peptidase 4 was not found to be expressed in taste buds, suggesting that the half - life of glp-1 in taste tissue should be high, ensuring sufficient concentrations within the taste bud to stimulate the glp-1 receptor . Hek-293 cells expressing g15 (a phospholipase c - linked g - protein), cotransfected with human and rat t1r2/t1r3, respond to all sweet taste stimuli: sucrose, fructose, galactose, glucose, lactose, and maltose . In the rat, however, the relative lack of t1r2 expression in taste bud cells of the fungiform papillae is consistent with the relative low response to sucrose recorded at the level of the chorda tympani nerve the activation of t1r1/t1r3 and t1r2/t1r3 in rat small intestine by glutamate, glucose, and artificial sweeteners increases the apical expression of glut2 and sugar absorption [83, 84]. Given this effect of sweet taste per se to activate t1r2/t1r3 and sugar absorption, it would have been expected that artificial sweeteners might also slow gastric emptying . However, intragastric or intestinal administration of equisweet solutions with artificial sweeteners and/or fructose did not modify glucose absorption rates, plasma glucose, incretin levels, or gastric emptying in humans [8587] or rodents as a glucose solution did . Collectively, these findings did not support the concept that the sweet taste per se is the principal detection mechanism, responsible for the regulation of gastric emptying, glucose absorption, or incretin release . Therefore, these data are in contrast to previously reported parallels between nutrient - sensing pathways in the oral cavity and gut . It seems that artificial sweeteners may influence the expression of sugar transporters such as glut2 [83, 84] and sglt1, but they may not influence other physiological functions such as gastric emptying or glucose absorption, and their effect on incretin release seems contradictory since some studies reported an effect of artificial sweeteners (sucralose) on glp-1 secretion from the ileum via t1r3 activation, while other studies did not find any effect of artificial sweeteners on incretin levels in humans [8587] or rodents when compared to glucose effects . Therefore, these data make unclear whether sweet taste receptors are necessary in such gastrointestinal functions . T1r1/t1r3 and t1r2/t1r3 stimulation leads to the activation of -gustducin . T1r3 and -gustducin are necessary for increased stimulation of (sglt1) by dietary sugars . T1r3 inhibition with lactisole decreased fructose stimulation of human sglt1, glut5, and l - pyruvate kinase mrna expression, demonstrating the implication of t1r3 in fructose signalling, whereas t1r3 did not control glut2 expression and activity . In enterocytes, cell polarity may influence the regulation of sweet taste receptor signalling . Tir2 and t1r3 are located at the basolateral membrane of differentiated enterocytes . Whereas the apical supply of fructose increased glut5 mrna expression, the basolateral supply of sugars increased glut2 expression, suggesting that sugars can directly signal from the basolateral membrane . When intestinal luminal glucose concentration is lower than in plasma, glucose is transported by sglt1 through the apical membrane against the concentration gradient . Dietary fructose is transported across the apical membrane by the facilitative transporter glut5 . In the basolateral membrane, both glucose and fructose are transported by glut2 [94, 95]. At high glucose or fructose concentrations, when sglt1 and glut5, respectively, are saturated, glut2 translocates to the apical membrane where it complements sglt1 and glut5 transport capacities . Depending on its relative abundance in the apical and basolateral membranes, it may stimulate sugar signals from intestinal lumen or bloodstream . Chronic exposure to a high - sugar diet promotes increased apical glut2 levels, increases glucose absorption, and excessive postprandial excursions . Insulin induces the internalisation of apical glut2, a process that is impaired in insulin resistance, contributing to further glucose absorption . Since glut2 depends on glucose transport by sglt1, i.e. It promotes glut2 upregulation, long - term regulation by sglt1 may also be reflected in changes in apical glut2 . In piglets fed isocaloric diets with variable concentrations of digestible carbohydrates (i.e. Sucrose and maize starch), sglt1 expression remains constant after exposure to diets containing up to 40% digestible carbohydrate . However, after exposure of> 50% carbohydrate diets, sglt1 expression is markedly increased . In contrast, under both low- and high - carbohydrate diets, glut2 is expressed on the basolateral membrane of pig enterocytes . These results suggest that sglt1 is the major route for the absorption of dietary sugars across the luminal membrane of swine enterocytes . Moreover, duodenal and jejunal infusions of glucose, fructose, and saccharin induced up - regulation of sglt1 in mice apparently involving vagal afferents . Altogether, these data suggest that sglt1 and apical glut2 are potential targets for antidiabetic therapy [90, 93, 94]. Duodenal sglt1 and glut5 mrna expressions and protein levels are substantially increased in diabetic patients . Reduction in plasma glucose in these patients promoted a reduction in both sglt1 and glut5 levels, suggesting that under hyperglycaemic conditions, the absorption of sugars is enhanced . Moreover, postprandial plasma fructose levels are increased in diabetic patients and are associated with the prevalence of diabetic retinopathy . In contrast, in zucker diabetic rats, mrna and protein levels of sglt1, glut5, and glut2 were unchanged compared to lean controls . This suggests that the zucker diabetic rat might not be a good model for the study of diabetes since it does not reproduce results observed in humans . Consumption of a large amount of pure fructose can exceed the capacity of intestinal fructose absorption, resulting in diarrhoea . However, the consumption of glucose along with fructose, as it is usually consumed in beverages and with meals (e.g. When provided as sucrose), appears to enhance fructose absorption . In addition, fructose absorption increases during sustained fructose consumption, suggesting an adaptation to increased fructose intake . Glut8, expressed only in the intracellular compartment, potentially mediates sugar transport into or out of intracellular organelles . Glut8 has high affinity for glucose, whereas fructose and galactose compete with glucose transport activity . Its deficiency enhances fructose uptake in cultured caco2 human intestinal epithelial cells and in jejunum isolated from mice lacking the gene encoding glut8 . Moreover, mice lacking glut8 rapidly develop higher serum fructose concentrations after oral fructose gavage . These effects are possibly mediated by the stabilisation of the dual - specificity glucose / fructose transporter glut12 . These data might lead to the speculation that this transporter could, in part, be implicated in fructose malabsorption previously reported when ingested at high levels in humans . The glucose - dependent secretion of glp-1 plays a critical role in the regulation of glucose homeostasis . It was shown that t1r3, but not t1r2, affects both incretin secretion from the intestine and insulin secretion from the pancreas . Exposure to glucose, fructose, and sucralose induced glp-1 secretion from the ileum of wild - type (t1r3) but not from t1r3 null mice (t1r3). T1r2 mice showed normal glycaemic control and partial small intestine glucose - stimulated glp-1 secretion, suggesting that t1r3 can mediate glucose - stimulated glp-1 secretion without t1r2 . Sglt1 and sglt3 may also be involved in enteric sugar - sensing and hormonal secretion stimulation . Sglt1 triggers glucose - induced secretion of gastrointestinal polypeptide (gip) from k - cells in the duodenum and jejunum . This in turn stimulates the release of glp-1 and glp-2 from l cells located in the ileum [107, 108]. Some evidence has revealed an anorexigenic effect of glucose and an orexigenic effect of fructose through different secretory profile of appetite peptides . For example, glucose and, to a lesser extent, galactose, but not fructose, mannose, or sorbitol, stimulated the release of gip . Glucose intragastrically infused or orally ingested induced an increase in plasma glucose levels, stimulated insulin, leptin, glp-1 and peptide tyrosine tyrosine (pyy) secretion, and reduced ghrelin secretion, while fructose did not substantially affect these hormones [37, 5759]. However, in one of these studies, the amounts of intragastric load of glucose and fructose were different (50 and 25 g/250 ml, respectively), which might have contributed to the observed differences . A possible explanation for the lack of effects of fructose on insulin secretion may be related to lower intestinal mrna and protein glut5 levels compared to glut2 levels, with the subsequent lower fructose transport compared to glucose . In addition, it was previously shown that glucose absorption rate was higher than that of fructose in yucatan minipigs . A substantial portion (12%) of ingested fructose is metabolised to lactate by the gut during absorption, while only 2% of glucose ingested is metabolised in the gut and almost all of the absorbed glucose appears in the portal vein as glucose . In concordance with lower fructose absorption rates compared to glucose, insulin concentrations were 7.5-fold above basal conditions following glucose intake, compared to threefold following fructose intake . Another possible explanation for the lack of effects of fructose on insulin secretion is the low level of expression of the glut5 fructose transporter in -cells . Taking together these data, i.e. Lower expression levels of glut5 than glut2 in the intestine, lower rate of fructose transport compared to glucose, partial intestinal fructose, but not glucose, metabolism, and the low level of glut5 expression in -cells could explain in part the lower increases in other gut peptides, besides insulin, induced by fructose ingestion, compared to glucose . Further studies are needed to confirm this concept . In this context, since insulin and leptin function as key signals in the central nervous system through the modulation of hypothalamic neuropeptides for the long - term regulation of energy balance, chronic fructose intake could lead to increased calorie intake, thereby contributing to weight gain and obesity . Hunger is regulated by the hypothalamus and the dorsal vagal complex in conjunction with an integrated network of limbic brain structures such as the striatum, orbitofrontal cortex, amygdala and insula, which control motivation - reward systems associated with the hedonic drive to eat . The arcuate nucleus of the hypothalamus is an integrator of hormonal and nutrient information to regulate both energy and glucose homeostasis . Brain cells are provided with mechanisms that sense energy availability in the extracellular space, such as an increase in adenosine monophosphate kinase (ampk) activity in response to an increase in amp - to - atp ratio . Glucose - sensing neurons are located in brain areas involved in the control of neuroendocrine function, nutrient metabolism, and energy homeostasis (e.g. Hypothalamic arcuate nucleus and the dorsal vagal complex) and also receive direct and indirect neural input from the periphery and from other brain areas that carry information about the characteristics of the ingested nutrients . Glucose - sensing neurons express receptors for and respond to peripheral hormones such as leptin and insulin that convey signals relating to carbohydrate and fat stores . These hormones as well as metabolic substances are transported across the bbb but can also freely diffuse from capillaries to the adjacent median eminence . Anabolic arcuate npy / agouti - related protein (agrp) and catabolic proopiomelanocortin (pomc) neurons are metabolic sensors with important roles as regulators of energy homeostasis . Arcuate npy / agrp neurons are inhibited by insulin and leptin and, when activated, stimulate food intake (orexigenic), whereas pomc neurons reduce food intake (anorexigenic) and are stimulated by insulin and leptin . Both neuronal subsets project to secondary order neurons located adjacent to hypothalamic areas including the paraventricular nucleus, where anorexigenic neurons are concentrated, and the lateral hypothalamic area, which contains orexigenic neurons . Npy / agrp neurons also inhibit pomc neurons via synaptic release of the inhibitory transmitter, -aminobutyric acid . Through smaller increases in insulin and leptin secretion induced by fructose intake, compared to glucose [55, 59], fructose - containing diets may lead to a lower inhibitory effect of orexigenic neurons npy / agrp, as well as a reduced reward value from food . Differential fuel utilisation responsible for the distinct responses of the npy / agrp and pomc neurons to metabolic signals has been characterised, whereas pomc neurons utilise glucose as the main fuel, npy / agrp neurons do not use glucose, but free fatty acids instead . This differential fuel utilisation implies two distinct and competitive mechanisms, glycolysis and - oxidation, in these neuronal populations . The by - products of substrate oxidation are reactive oxygen species (ros) that have a crucial role in the acute and the long - term regulation of feeding, satiety, and associated metabolic changes (i.e. Glucose and fatty acid homeostasis). Mitochondrial ros (mros) is a necessary signal to initiate the response to glucose sensing . A finely controlled mros production might be considered as an essential physiological messenger in metabolic - sensitive cells [114, 115]. Alteration of the hypothalamic glucose - sensing mechanism induced dramatic effects on energy balance correlated to abnormal redox signalling originated from mitochondrial dysfunction . During negative energy balance, npy / agrp neurons utilise free fatty acids as fuel, but ros levels are not increased in these cells despite increased firing and substrate utilisation . In contrast, during positive energy balance, when glucose - utilising pomc neurons are firing at high levels, ros accumulate in these pomc cells because they do not need to be buffered . Sustained the fact that satiety is associated with the highest levels of ros production in the pomc neurons indicates that these cells are more exposed to ros - induced damage than npy / agrp neurons, which do not produce elevated ros levels even if highly active . Thus, it seems plausible that pomc neurons are more exposed to elevated firing (positive energy balance) over time, thus leading to pomc system impairment . In contrast, since npy / agrp neurons are inherently able to buffer ros, their increased activity during negative energy balance is not associated with ros - induced degeneration . Regulatory mechanisms of food intake controlled by central ampk activity in response to an i.c.v . Glucose injection through inactivation of ampk, an increase in malonyl coa, and in anorexigenic neuropeptides mrna levels in the hypothalamus, whereas i.c.v . Fructose injection resulted in the inverse effects [50, 54, 117, 118] (see fig . 1). However, whether fructose orally ingested can also produce these effects than when administered i.p . Or fructose bypasses the rate - limiting step in glycolysis, which generates a decrease in amp / atp ratio, the phosphorylation and activation of ampk (the cell sensor of amp / atp ratio) in the liver, and in hypothalamic neurons [50, 54]. This stimulates corticosterone secretion, activating glucocorticoid receptors followed by activation of phosphoenol pyruvate carboxykinase (pepck) and gluconeogenesis . The activation of pepck induced by fructose was prevented by ru486, a glucocorticoid receptor antagonist . Intracerebroventricular (i.c.v) injection of the glp-1 receptor (glp-1r) agonist exendin-4 (ex-4), suppressed ampk activity in hypothalamic cells and food intake; i.c.v fructose attenuated the anorectic effect of ex-4, suggesting a mechanism for the increased food intake by fructose via impairment of central glp-1r action . Glucose injected i.c.v . Increased atp / amp ratio, activated ampk, acetyl - coa carboxylase (acc) and malonyl coa, leading to decreased mrna levels of orexigenic neuropeptides npy and agouti - related protein (agrp), while activating the expression of the anorexigenic peptides cocaine amphetamine - related transcript (cart) and proopiomelanocortin (pomc). Fructose i.c.v injected exerts an orexigenic effect by lowering malonyl coa mrna levels effects of an i.c.v . Fructose bypasses the rate - limiting step in glycolysis, which generates a decrease in amp / atp ratio, the phosphorylation and activation of ampk (the cell sensor of amp / atp ratio) in the liver, and in hypothalamic neurons [50, 54]. This stimulates corticosterone secretion, activating glucocorticoid receptors followed by activation of phosphoenol pyruvate carboxykinase (pepck) and gluconeogenesis . The activation of pepck induced by fructose was prevented by ru486, a glucocorticoid receptor antagonist . Intracerebroventricular (i.c.v) injection of the glp-1 receptor (glp-1r) agonist exendin-4 (ex-4), suppressed ampk activity in hypothalamic cells and food intake; i.c.v fructose attenuated the anorectic effect of ex-4, suggesting a mechanism for the increased food intake by fructose via impairment of central glp-1r action . Glucose injected i.c.v . Increased atp / amp ratio, activated ampk, acetyl - coa carboxylase (acc) and malonyl coa, leading to decreased mrna levels of orexigenic neuropeptides npy and agouti - related protein (agrp), while activating the expression of the anorexigenic peptides cocaine amphetamine - related transcript (cart) and proopiomelanocortin (pomc). Fructose i.c.v injected exerts an orexigenic effect by lowering malonyl coa mrna levels another potential mechanism for the fructose effects on food intake might be through the nuclear receptors liver x receptor (lxr) and . These receptors have been previously implicated in the regulation of carbohydrate and lipid metabolism and were shown to be expressed in the hypothalamus and implicated in the regulation of food intake . Free access to a diet - containing 10% fructose for 6 weeks in glucose - intolerant rats induced a decrease in lxr and an increase in lxr in the hypothalamus, but not in the hippocampus, cerebellum, or neocortex . It is possible that the specific hypothalamic increase in lxr by fructose may trigger neurochemical and neurophysiological responses for the control of food intake and energy expenditure . But circulating fructose levels could possibly promote central effects in humans even if hepatic clearance of fructose is extremely efficient, given the following observations: (i) the presence of glut5 in the bbb and ketohexokinase mrna, the necessary cellular machinery for fructose metabolism; (ii) fructose administered i.p . Can cross the bbb and trigger neuronal activation in rodents; (iii) fructose administrated i.p . These data suggested the capacity of fructose to cross the bbb into the hypothalamus, where it could be metabolised and used as an energy source . Thus, consumption of high - fructose diets might probably have a direct effect on the brain, but no study has clearly proven this concept yet . While it is well established that glucose orally ingested undergoes facilitated transport across the bbb, the demonstration that fructose orally ingested can cross the bbb is still missing . Another question is how changes in feeding behaviour associated with glucose- and fructose - induced activation of brain regions observed in animals could be extrapolated to humans, especially when most of the studies investigating these effects have been performed on rodents . New technologies are available to facilitate the translation of animal to human studies and help understanding of brain functions . One such technique is the single - photon emission computed tomography (spect) that provides a way to compare brain circuits implicated in the processing of oral and/or visceral (e.g. Duodenal or portal) sugar signals . The authors found that both duodenal and portal glucose infusions activated the dorsolateral prefrontal cortex and primary somatosensory cortex . However, only duodenal glucose infusion induced the activation of the prepyriform area, orbitofrontal cortex, caudate and putamen, and the deactivation of the anterior prefrontal cortex and anterior entorhinal cortex, whereas only portal glucose infusion induced the activation of the insular cortex . These results indicated that duodenal and portal glucose infusions modulate differentially the activity of brain areas implicated in the regulation of eating behaviour, which probably explains the decrease in food intake after both stimulations . Another spect study in pigs (clouard et al ., 2013, unpublished data) demonstrated that combined oral and duodenal sucrose sensing induced activation of brain regions involved in memory, reward processes, and hedonic identification of sensory stimuli (i.e. Amygdala, dorsal striatum: caudate and putamen, and the anterior prefrontal cortex), whereas oral or duodenal sucrose sensing individually administered did not . These findings suggested that (1) the concordance between oral and visceral signals (sweet taste and calories) during sugar sensing is necessary for the onset of responses in these brain structures, or (2) the synergy between oral and visceral signals during sugar sensing is required to obtain a signal that is strong enough to trigger brain responses in these structures (clouard, et al . Positron emission tomography (pet) studies with 18-fluorodeoxyglucose (18-fdg) to measure brain glucose metabolism in normal - weight individuals reported that exposure to food cues increased metabolic activity in the orbitofrontal cortex, similar to that observed in cocaine - addicted subjects, which was an effect associated with the perception of hunger and the desire for food . Functional magnetic resonance imaging (fmri) is another technique that provides a non - invasive way to assess the effects of glucose and fructose intake, as well as of obesity, on regional cerebral blood flow (abbreviated rcbf and estimated via the blood oxygen - level - dependent or bold signal). Previous studies have explored the temporal response to glucose intake or infusion using fmri and found suppression of hypothalamic bold signalling after the administration of glucose to rats and humans . Obese subjects presented diminished attenuation of the bold signal in response to glucose ingestion compared with lean subjects, and patients with type 2 diabetes did not show any hypothalamic signal changes compared with non - diabetic patients . This might suggest a reduced neuronal activation in obese relative to lean subjects, which might translate in no suppression of appetite and less rewarding signals from glucose intake leading to overeating . Cortical responses to sugars in healthy subjects as assessed by fmri appear to be opposite between glucose and fructose infusion: increased and decreased cortical activation, respectively . The suppressive effect of fructose on cortical bold signal occurred despite the fact that cortical - specific receptors (glut5) are present in low concentrations throughout the brain, where the glucose transporter glut3 predominates . Whether this cortical response to fructose is due to effects mediated by glut2 and glut5 carriers in the bbb or in local glial cells, due to increased osmolality, or is the indirect result of changes in the levels of peripheral neural input or metabolic intermediaries is yet to be understood . However, the overall observations suggest a major implication of fructose on changes in brain activity similar to those observed with addictive drugs, which may lead to an altered reward response to palatable food . In contrast to the effects of sugars in cortical activation, their effects in other brain regions involved in food intake control, i.e. Hypothalamus, appear to be opposite . Another fmri study in humans showed that glucose, but not fructose intake, induced a marked reduction in hypothalamic bold signal, as well as a reduction in cbf within the thalamus, insula, anterior cingulate, striatum, and hypothalamus, i.e. Brain regions that act together to sense the metabolic state of an individual and drive motivation and reward . Moreover, fructose produced a transient increase in hypothalamic activity and reduced cbf in the hippocampus, a region implicated not only in memory but also influencing emotional responses to food intake . These findings suggest that ingestion of glucose, but not fructose, initiates a coordinated response between the homeostatic and striatal networks that regulate eating behaviour . In line with these data, ingestion of glucose but what are the underlying mechanisms of these effects of fructose on cbf changes is a remaining question since no study has clearly demonstrated the capacity of fructose orally ingested to cross the bbb . Figures 2 and 3 represent hypothetical and controversial models summarising the findings that support the fructose hypothesis which postulates an orexigenic and less rewarding effect of fructose intake, compared to glucose . It presents the underlying molecular mechanisms throughout the gut brain communication supporting this hypothesis.fig . Vagal afferents express glp-1 and 5-ht receptors, and are implicated in the regulation of insulin secretion . Many neuronal signals are communicated via the vagus nerve to the brain stem, which relays the glucose signal to hypothalamic nuclei and then to the pertinent target cells: npy / agrp and pomc / cart neurons . Npy neuropeptide y, agrp agouti - related protein, pomc proopiomelanocortin, cart cocaine amphetamine - related transcript, amp adenosine monophosphate, ampk amp kinase, acc acetyl - coa carboxylase . Black arrows activation; discontinued red lines inhibition; thin discontinued red line weak activation [4, 51, 52, 54, 57, 58, 72, 182]fig . Luminal fructose induces weak release of 5-ht and glp-1 from enteroendocrine and l cells, respectively, weak pyy, insulin and leptin secretion, as well as weak ghrelin suppression . Many neuronal signals are communicated via the vagus nerve to the brain stem, which relays the glucose signal to hypothalamic nuclei and then to the pertinent target cells: npy / agrp and pomc / cart neurons . Npy neuropeptide y, agrp agouti - related protein, pomc proopiomelanocortin, cart cocaine amphetamine - related transcript, amp adenosine monophosphate, ampk amp kinase, acc acetyl - coa carboxylase . Black arrows activation; discontinued red lines inhibition; grey arrows low secretion or low activation [4, 37, 51, 52, 54, 58, 59, 72, 182] hypothetical model of the peripheral and central effects of glucose on food intake . Vagal afferents express glp-1 and 5-ht receptors, and are implicated in the regulation of insulin secretion . Many neuronal signals are communicated via the vagus nerve to the brain stem, which relays the glucose signal to hypothalamic nuclei and then to the pertinent target cells: npy / agrp and pomc / cart neurons . Npy neuropeptide y, agrp agouti - related protein, pomc proopiomelanocortin, cart cocaine amphetamine - related transcript, amp adenosine monophosphate, ampk amp kinase, acc acetyl - coa carboxylase . Black arrows activation; discontinued red lines inhibition; thin discontinued red line weak activation [4, 51, 52, 54, 57, 58, 72, 182] hypothetical model of the peripheral and central effects of fructose on food intake . Luminal fructose induces weak release of 5-ht and glp-1 from enteroendocrine and l cells, respectively, weak pyy, insulin and leptin secretion, as well as weak ghrelin suppression . Many neuronal signals are communicated via the vagus nerve to the brain stem, which relays the glucose signal to hypothalamic nuclei and then to the pertinent target cells: npy / agrp and pomc / cart neurons . Npy neuropeptide y, agrp agouti - related protein, pomc proopiomelanocortin, cart cocaine amphetamine - related transcript, amp adenosine monophosphate, ampk amp kinase, acc acetyl - coa carboxylase . Black arrows activation; discontinued red lines inhibition; grey arrows low secretion or low activation [4, 37, 51, 52, 54, 58, 59, 72, 182] peripheral sweet taste and sugar detectors are key regulators of feeding behaviour and energy homeostasis . Taste - signalling mechanisms identified in the oral epithelium also operate in the gut and play a role in both sugar detection and regulation of intestinal and pancreatic hormone secretion . There are two main groups of sugar detectors: members of the g - protein - coupled receptors (gpcr) family, and sugar transporters (e.g. Glut2, glut5, sodium - dependent glucose co - transporter 1 (sglt1), and glut8). Enteroendocrine cells directly sense sugars via gpcr, including the sweet taste receptors of type 1, t1r . The initial step in taste recognition occurs on the apical surface of taste receptor cells, within taste buds of the tongue and palate . The regulation of taste sensitivity by appetite peptides at the level of taste bud cells in the tongue as well as in enteroendocrine cells of the taste epithelium may be important in the control of eating behaviour and the regulation of energy homeostasis . However, this concept remains unclear since it was found that knockout (ko) mice (p2x2/p2x3) with adenosine triphosphate taste cell signalling deficits show relatively normal food intake and body weight . They also develop strong preferences for non - taste nutrients by the post - oral actions of these nutrients . Furthermore, much of the research on appetite peptides and taste detection in the mouth has been performed at the cell / neuron level; however, little empirical evidence exists to date for these peptides impacting taste function at the level of the mouth . Therefore, more studies should be performed in large animal models to clarify these concepts . Appetite regulatory peptides, such as leptin, endocannabinoids, glp-1, glucagon, oxytocin, insulin, cholecystokinin (cck), neuropeptide y (npy), and vasoactive intestinal peptide (vip), modulate taste sensitivity at the level of oral sweet taste cells . Leptin selectively suppressed sweet taste responses of cells isolated from circumvallate papillae from non - diabetic mice, but not in diabetic db / db mice . This indicated that the effect of leptin on sweet taste sensitivity is mediated by the leptin receptor expressed in these cells . The t1r3 subunit is co - expressed with the leptin receptor in both fungiform and circumvallate taste bud cells, and leptin suppresses sweet taste sensitivity in mice by affecting responsiveness of t1r3-expressing taste cells via the leptin receptor . Therefore, leptin may play an important role in the regulation of sweet taste sensitivity in the tongue, besides its central actions on food intake . Co - expression of glp-1 with taste - signalling elements such as t1r2, t1r3, and -gustducin, a gi family member associated with taste perception, was found in human intestinal endocrine l cells . These taste - signalling elements mediate the glucose - dependent secretion of glp-1 and maintain or enhance sweet taste sensitivity via paracrine action . In addition to its intestinal expression, glp-1 was also found to be expressed in taste cells in mouse circumvallate papillae taste buds; it was co - expressed with -gustducin and t1r3-expressing sweet taste cells in mouse taste buds, and it was produced in taste buds from lingual extracts in its active form . In contrast to its presence in blood and ileum, dipeptidyl peptidase 4 was not found to be expressed in taste buds, suggesting that the half - life of glp-1 in taste tissue should be high, ensuring sufficient concentrations within the taste bud to stimulate the glp-1 receptor . Hek-293 cells expressing g15 (a phospholipase c - linked g - protein), cotransfected with human and rat t1r2/t1r3, respond to all sweet taste stimuli: sucrose, fructose, galactose, glucose, lactose, and maltose . In the rat, however, the relative lack of t1r2 expression in taste bud cells of the fungiform papillae is consistent with the relative low response to sucrose recorded at the level of the chorda tympani nerve the activation of t1r1/t1r3 and t1r2/t1r3 in rat small intestine by glutamate, glucose, and artificial sweeteners increases the apical expression of glut2 and sugar absorption [83, 84]. Given this effect of sweet taste per se to activate t1r2/t1r3 and sugar absorption, it would have been expected that artificial sweeteners might also slow gastric emptying . However, intragastric or intestinal administration of equisweet solutions with artificial sweeteners and/or fructose did not modify glucose absorption rates, plasma glucose, incretin levels, or gastric emptying in humans [8587] or rodents as a glucose solution did . Collectively, these findings did not support the concept that the sweet taste per se is the principal detection mechanism, responsible for the regulation of gastric emptying, glucose absorption, or incretin release . Therefore, these data are in contrast to previously reported parallels between nutrient - sensing pathways in the oral cavity and gut . It seems that artificial sweeteners may influence the expression of sugar transporters such as glut2 [83, 84] and sglt1, but they may not influence other physiological functions such as gastric emptying or glucose absorption, and their effect on incretin release seems contradictory since some studies reported an effect of artificial sweeteners (sucralose) on glp-1 secretion from the ileum via t1r3 activation, while other studies did not find any effect of artificial sweeteners on incretin levels in humans [8587] or rodents when compared to glucose effects . Therefore, these data make unclear whether sweet taste receptors are necessary in such gastrointestinal functions . T1r1/t1r3 and t1r2/t1r3 stimulation leads to the activation of -gustducin . T1r3 and -gustducin are necessary for increased stimulation of (sglt1) by dietary sugars . T1r3 inhibition with lactisole decreased fructose stimulation of human sglt1, glut5, and l - pyruvate kinase mrna expression, demonstrating the implication of t1r3 in fructose signalling, whereas t1r3 did not control glut2 expression and activity . In enterocytes, tir2 and t1r3 are located at the basolateral membrane of differentiated enterocytes . Whereas the apical supply of fructose increased glut5 mrna expression, the basolateral supply of sugars increased glut2 expression, suggesting that sugars can directly signal from the basolateral membrane . When intestinal luminal glucose concentration is lower than in plasma, glucose is transported by sglt1 through the apical membrane against the concentration gradient . Dietary fructose is transported across the apical membrane by the facilitative transporter glut5 . In the basolateral membrane, both glucose and fructose are transported by glut2 [94, 95]. At high glucose or fructose concentrations, when sglt1 and glut5, respectively, are saturated, glut2 translocates to the apical membrane where it complements sglt1 and glut5 transport capacities . Depending on its relative abundance in the apical and basolateral membranes, it may stimulate sugar signals from intestinal lumen or bloodstream . Chronic exposure to a high - sugar diet promotes increased apical glut2 levels, increases glucose absorption, and excessive postprandial excursions . Insulin induces the internalisation of apical glut2, a process that is impaired in insulin resistance, contributing to further glucose absorption . Since glut2 depends on glucose transport by sglt1, i.e. It promotes glut2 upregulation, long - term regulation by sglt1 may also be reflected in changes in apical glut2 . In piglets fed isocaloric diets with variable concentrations of digestible carbohydrates (i.e. Sucrose and maize starch), sglt1 expression remains constant after exposure to diets containing up to 40% digestible carbohydrate . However, after exposure of> 50% carbohydrate diets, sglt1 expression is markedly increased . In contrast, under both low- and high - carbohydrate diets, glut2 is expressed on the basolateral membrane of pig enterocytes . These results suggest that sglt1 is the major route for the absorption of dietary sugars across the luminal membrane of swine enterocytes . Moreover, duodenal and jejunal infusions of glucose, fructose, and saccharin induced up - regulation of sglt1 in mice apparently involving vagal afferents . Altogether, these data suggest that sglt1 and apical glut2 are potential targets for antidiabetic therapy [90, 93, 94]. Duodenal sglt1 and glut5 mrna expressions and protein levels are substantially increased in diabetic patients . Reduction in plasma glucose in these patients promoted a reduction in both sglt1 and glut5 levels, suggesting that under hyperglycaemic conditions, the absorption of sugars is enhanced . Moreover, postprandial plasma fructose levels are increased in diabetic patients and are associated with the prevalence of diabetic retinopathy . In contrast, in zucker diabetic rats, mrna and protein levels of sglt1, glut5, and glut2 were unchanged compared to lean controls . This suggests that the zucker diabetic rat might not be a good model for the study of diabetes since it does not reproduce results observed in humans . Consumption of a large amount of pure fructose can exceed the capacity of intestinal fructose absorption, resulting in diarrhoea . However, the consumption of glucose along with fructose, as it is usually consumed in beverages and with meals (e.g. When provided as sucrose), appears to enhance fructose absorption . In addition, fructose absorption increases during sustained fructose consumption, suggesting an adaptation to increased fructose intake . Glut8, expressed only in the intracellular compartment, potentially mediates sugar transport into or out of intracellular organelles . Glut8 has high affinity for glucose, whereas fructose and galactose compete with glucose transport activity . Its deficiency enhances fructose uptake in cultured caco2 human intestinal epithelial cells and in jejunum isolated from mice lacking the gene encoding glut8 . Moreover, mice lacking glut8 rapidly develop higher serum fructose concentrations after oral fructose gavage . These effects are possibly mediated by the stabilisation of the dual - specificity glucose / fructose transporter glut12 . These data might lead to the speculation that this transporter could, in part, be implicated in fructose malabsorption previously reported when ingested at high levels in humans . The glucose - dependent secretion of glp-1 plays a critical role in the regulation of glucose homeostasis . It was shown that t1r3, but not t1r2, affects both incretin secretion from the intestine and insulin secretion from the pancreas . Exposure to glucose, fructose, and sucralose induced glp-1 secretion from the ileum of wild - type (t1r3) but not from t1r3 null mice (t1r3). T1r2 mice showed normal glycaemic control and partial small intestine glucose - stimulated glp-1 secretion, suggesting that t1r3 can mediate glucose - stimulated glp-1 secretion without t1r2 . Sglt1 and sglt3 may also be involved in enteric sugar - sensing and hormonal secretion stimulation . Sglt1 triggers glucose - induced secretion of gastrointestinal polypeptide (gip) from k - cells in the duodenum and jejunum . This in turn stimulates the release of glp-1 and glp-2 from l cells located in the ileum [107, 108]. Some evidence has revealed an anorexigenic effect of glucose and an orexigenic effect of fructose through different secretory profile of appetite peptides . For example, glucose and, to a lesser extent, galactose, but not fructose, mannose, or sorbitol, stimulated the release of gip . Glucose intragastrically infused or orally ingested induced an increase in plasma glucose levels, stimulated insulin, leptin, glp-1 and peptide tyrosine tyrosine (pyy) secretion, and reduced ghrelin secretion, while fructose did not substantially affect these hormones [37, 5759]. However, in one of these studies, the amounts of intragastric load of glucose and fructose were different (50 and 25 g/250 ml, respectively), which might have contributed to the observed differences . A possible explanation for the lack of effects of fructose on insulin secretion may be related to lower intestinal mrna and protein glut5 levels compared to glut2 levels, with the subsequent lower fructose transport compared to glucose . In addition, it was previously shown that glucose absorption rate was higher than that of fructose in yucatan minipigs . A substantial portion (12%) of ingested fructose is metabolised to lactate by the gut during absorption, while only 2% of glucose ingested is metabolised in the gut and almost all of the absorbed glucose appears in the portal vein as glucose . In concordance with lower fructose absorption rates compared to glucose, insulin concentrations were 7.5-fold above basal conditions following glucose intake, compared to threefold following fructose intake . Another possible explanation for the lack of effects of fructose on insulin secretion is the low level of expression of the glut5 fructose transporter in -cells . Taking together these data, i.e. Lower expression levels of glut5 than glut2 in the intestine, lower rate of fructose transport compared to glucose, partial intestinal fructose, but not glucose, metabolism, and the low level of glut5 expression in -cells could explain in part the lower increases in other gut peptides, besides insulin, induced by fructose ingestion, compared to glucose . Further studies are needed to confirm this concept . In this context, since insulin and leptin function as key signals in the central nervous system through the modulation of hypothalamic neuropeptides for the long - term regulation of energy balance, chronic fructose intake could lead to increased calorie intake, thereby contributing to weight gain and obesity . Hunger is regulated by the hypothalamus and the dorsal vagal complex in conjunction with an integrated network of limbic brain structures such as the striatum, orbitofrontal cortex, amygdala and insula, which control motivation - reward systems associated with the hedonic drive to eat . The arcuate nucleus of the hypothalamus is an integrator of hormonal and nutrient information to regulate both energy and glucose homeostasis . Brain cells are provided with mechanisms that sense energy availability in the extracellular space, such as an increase in adenosine monophosphate kinase (ampk) activity in response to an increase in amp - to - atp ratio . Glucose - sensing neurons are located in brain areas involved in the control of neuroendocrine function, nutrient metabolism, and energy homeostasis (e.g. Hypothalamic arcuate nucleus and the dorsal vagal complex) and also receive direct and indirect neural input from the periphery and from other brain areas that carry information about the characteristics of the ingested nutrients . Glucose - sensing neurons express receptors for and respond to peripheral hormones such as leptin and insulin that convey signals relating to carbohydrate and fat stores . These hormones as well as metabolic substances are transported across the bbb but can also freely diffuse from capillaries to the adjacent median eminence . Anabolic arcuate npy / agouti - related protein (agrp) and catabolic proopiomelanocortin (pomc) neurons are metabolic sensors with important roles as regulators of energy homeostasis . Arcuate npy / agrp neurons are inhibited by insulin and leptin and, when activated, stimulate food intake (orexigenic), whereas pomc neurons reduce food intake (anorexigenic) and are stimulated by insulin and leptin . Both neuronal subsets project to secondary order neurons located adjacent to hypothalamic areas including the paraventricular nucleus, where anorexigenic neurons are concentrated, and the lateral hypothalamic area, which contains orexigenic neurons . Npy / agrp neurons also inhibit pomc neurons via synaptic release of the inhibitory transmitter, -aminobutyric acid . Through smaller increases in insulin and leptin secretion induced by fructose intake, compared to glucose [55, 59], fructose - containing diets may lead to a lower inhibitory effect of orexigenic neurons npy / agrp, as well as a reduced reward value from food . Differential fuel utilisation responsible for the distinct responses of the npy / agrp and pomc neurons to metabolic signals has been characterised, whereas pomc neurons utilise glucose as the main fuel, npy / agrp neurons do not use glucose, but free fatty acids instead . This differential fuel utilisation implies two distinct and competitive mechanisms, glycolysis and - oxidation, in these neuronal populations . When glycolysis is elevated, - oxidation is inhibited and vice versa . The by - products of substrate oxidation are reactive oxygen species (ros) that have a crucial role in the acute and the long - term regulation of feeding, satiety, and associated metabolic changes (i.e. Glucose and fatty acid homeostasis). Mitochondrial ros (mros) is a necessary signal to initiate the response to glucose sensing . A finely controlled mros production might be considered as an essential physiological messenger in metabolic - sensitive cells [114, 115]. Alteration of the hypothalamic glucose - sensing mechanism induced dramatic effects on energy balance correlated to abnormal redox signalling originated from mitochondrial dysfunction . During negative energy balance, npy / agrp neurons utilise free fatty acids as fuel, but ros levels are not increased in these cells despite increased firing and substrate utilisation . In contrast, during positive energy balance, when glucose - utilising pomc neurons are firing at high levels, ros accumulate in these pomc cells because they do not need to be buffered . Sustained the fact that satiety is associated with the highest levels of ros production in the pomc neurons indicates that these cells are more exposed to ros - induced damage than npy / agrp neurons, which do not produce elevated ros levels even if highly active . Thus, it seems plausible that pomc neurons are more exposed to elevated firing (positive energy balance) over time, thus leading to pomc system impairment . In contrast, since npy / agrp neurons are inherently able to buffer ros, their increased activity during negative energy balance is not associated with ros - induced degeneration . Regulatory mechanisms of food intake controlled by central ampk activity in response to an i.c.v . Glucose injection through inactivation of ampk, an increase in malonyl coa, and in anorexigenic neuropeptides mrna levels in the hypothalamus, whereas i.c.v . Fructose injection resulted in the inverse effects [50, 54, 117, 118] (see fig . 1). However, whether fructose orally ingested can also produce these effects than when administered i.p . Or fructose bypasses the rate - limiting step in glycolysis, which generates a decrease in amp / atp ratio, the phosphorylation and activation of ampk (the cell sensor of amp / atp ratio) in the liver, and in hypothalamic neurons [50, 54]. This stimulates corticosterone secretion, activating glucocorticoid receptors followed by activation of phosphoenol pyruvate carboxykinase (pepck) and gluconeogenesis . The activation of pepck induced by fructose was prevented by ru486, a glucocorticoid receptor antagonist . Intracerebroventricular (i.c.v) injection of the glp-1 receptor (glp-1r) agonist exendin-4 (ex-4), suppressed ampk activity in hypothalamic cells and food intake; i.c.v fructose attenuated the anorectic effect of ex-4, suggesting a mechanism for the increased food intake by fructose via impairment of central glp-1r action . Glucose injected i.c.v . Increased atp / amp ratio, activated ampk, acetyl - coa carboxylase (acc) and malonyl coa, leading to decreased mrna levels of orexigenic neuropeptides npy and agouti - related protein (agrp), while activating the expression of the anorexigenic peptides cocaine amphetamine - related transcript (cart) and proopiomelanocortin (pomc). Fructose i.c.v injected exerts an orexigenic effect by lowering malonyl coa mrna levels effects of an i.c.v . Fructose bypasses the rate - limiting step in glycolysis, which generates a decrease in amp / atp ratio, the phosphorylation and activation of ampk (the cell sensor of amp / atp ratio) in the liver, and in hypothalamic neurons [50, 54]. This stimulates corticosterone secretion, activating glucocorticoid receptors followed by activation of phosphoenol pyruvate carboxykinase (pepck) and gluconeogenesis . The activation of pepck induced by fructose was prevented by ru486, a glucocorticoid receptor antagonist . Intracerebroventricular (i.c.v) injection of the glp-1 receptor (glp-1r) agonist exendin-4 (ex-4), suppressed ampk activity in hypothalamic cells and food intake; i.c.v fructose attenuated the anorectic effect of ex-4, suggesting a mechanism for the increased food intake by fructose via impairment of central glp-1r action . Glucose injected i.c.v . Increased atp / amp ratio, activated ampk, acetyl - coa carboxylase (acc) and malonyl coa, leading to decreased mrna levels of orexigenic neuropeptides npy and agouti - related protein (agrp), while activating the expression of the anorexigenic peptides cocaine amphetamine - related transcript (cart) and proopiomelanocortin (pomc). Fructose i.c.v injected exerts an orexigenic effect by lowering malonyl coa mrna levels another potential mechanism for the fructose effects on food intake might be through the nuclear receptors liver x receptor (lxr) and . These receptors have been previously implicated in the regulation of carbohydrate and lipid metabolism and were shown to be expressed in the hypothalamus and implicated in the regulation of food intake . Free access to a diet - containing 10% fructose for 6 weeks in glucose - intolerant rats induced a decrease in lxr and an increase in lxr in the hypothalamus, but not in the hippocampus, cerebellum, or neocortex . It is possible that the specific hypothalamic increase in lxr by fructose may trigger neurochemical and neurophysiological responses for the control of food intake and energy expenditure . But circulating fructose levels could possibly promote central effects in humans even if hepatic clearance of fructose is extremely efficient, given the following observations: (i) the presence of glut5 in the bbb and ketohexokinase mrna, the necessary cellular machinery for fructose metabolism; (ii) fructose administered i.p . Can cross the bbb and trigger neuronal activation in rodents; (iii) fructose administrated i.p . These data suggested the capacity of fructose to cross the bbb into the hypothalamus, where it could be metabolised and used as an energy source . Thus, consumption of high - fructose diets might probably have a direct effect on the brain, but no study has clearly proven this concept yet . While it is well established that glucose orally ingested undergoes facilitated transport across the bbb, the demonstration that fructose orally ingested can cross the bbb is still missing . Another question is how changes in feeding behaviour associated with glucose- and fructose - induced activation of brain regions observed in animals could be extrapolated to humans, especially when most of the studies investigating these effects have been performed on rodents . New technologies are available to facilitate the translation of animal to human studies and help understanding of brain functions . One such technique is the single - photon emission computed tomography (spect) that provides a way to compare brain circuits implicated in the processing of oral and/or visceral (e.g. Duodenal or portal) sugar signals . The authors found that both duodenal and portal glucose infusions activated the dorsolateral prefrontal cortex and primary somatosensory cortex . However, only duodenal glucose infusion induced the activation of the prepyriform area, orbitofrontal cortex, caudate and putamen, and the deactivation of the anterior prefrontal cortex and anterior entorhinal cortex, whereas only portal glucose infusion induced the activation of the insular cortex . These results indicated that duodenal and portal glucose infusions modulate differentially the activity of brain areas implicated in the regulation of eating behaviour, which probably explains the decrease in food intake after both stimulations . Another spect study in pigs (clouard et al ., 2013, unpublished data) demonstrated that combined oral and duodenal sucrose sensing induced activation of brain regions involved in memory, reward processes, and hedonic identification of sensory stimuli (i.e. Amygdala, dorsal striatum: caudate and putamen, and the anterior prefrontal cortex), whereas oral or duodenal sucrose sensing individually administered did not . These findings suggested that (1) the concordance between oral and visceral signals (sweet taste and calories) during sugar sensing is necessary for the onset of responses in these brain structures, or (2) the synergy between oral and visceral signals during sugar sensing is required to obtain a signal that is strong enough to trigger brain responses in these structures (clouard, et al . Positron emission tomography (pet) studies with 18-fluorodeoxyglucose (18-fdg) to measure brain glucose metabolism in normal - weight individuals reported that exposure to food cues increased metabolic activity in the orbitofrontal cortex, similar to that observed in cocaine - addicted subjects, which was an effect associated with the perception of hunger and the desire for food . Functional magnetic resonance imaging (fmri) is another technique that provides a non - invasive way to assess the effects of glucose and fructose intake, as well as of obesity, on regional cerebral blood flow (abbreviated rcbf and estimated via the blood oxygen - level - dependent or bold signal). Previous studies have explored the temporal response to glucose intake or infusion using fmri and found suppression of hypothalamic bold signalling after the administration of glucose to rats and humans . Obese subjects presented diminished attenuation of the bold signal in response to glucose ingestion compared with lean subjects, and patients with type 2 diabetes did not show any hypothalamic signal changes compared with non - diabetic patients . This might suggest a reduced neuronal activation in obese relative to lean subjects, which might translate in no suppression of appetite and less rewarding signals from glucose intake leading to overeating . Cortical responses to sugars in healthy subjects as assessed by fmri appear to be opposite between glucose and fructose infusion: increased and decreased cortical activation, respectively . The suppressive effect of fructose on cortical bold signal occurred despite the fact that cortical - specific receptors (glut5) are present in low concentrations throughout the brain, where the glucose transporter glut3 predominates . Whether this cortical response to fructose is due to effects mediated by glut2 and glut5 carriers in the bbb or in local glial cells, due to increased osmolality, or is the indirect result of changes in the levels of peripheral neural input or metabolic intermediaries is yet to be understood . However, the overall observations suggest a major implication of fructose on changes in brain activity similar to those observed with addictive drugs, which may lead to an altered reward response to palatable food . In contrast to the effects of sugars in cortical activation, their effects in other brain regions involved in food intake control, i.e. Hypothalamus, appear to be opposite . Another fmri study in humans showed that glucose, but not fructose intake, induced a marked reduction in hypothalamic bold signal, as well as a reduction in cbf within the thalamus, insula, anterior cingulate, striatum, and hypothalamus, i.e. Brain regions that act together to sense the metabolic state of an individual and drive motivation and reward . Moreover, fructose produced a transient increase in hypothalamic activity and reduced cbf in the hippocampus, a region implicated not only in memory but also influencing emotional responses to food intake . These findings suggest that ingestion of glucose, but not fructose, initiates a coordinated response between the homeostatic and striatal networks that regulate eating behaviour . In line with these data, ingestion of glucose but not fructose produced increased ratings of satiety and fullness . What are the underlying mechanisms of these effects of fructose on cbf changes is a remaining question since no study has clearly demonstrated the capacity of fructose orally ingested to cross the bbb . Figures 2 and 3 represent hypothetical and controversial models summarising the findings that support the fructose hypothesis which postulates an orexigenic and less rewarding effect of fructose intake, compared to glucose . Vagal afferents express glp-1 and 5-ht receptors, and are implicated in the regulation of insulin secretion . Many neuronal signals are communicated via the vagus nerve to the brain stem, which relays the glucose signal to hypothalamic nuclei and then to the pertinent target cells: npy / agrp and pomc / cart neurons . Npy neuropeptide y, agrp agouti - related protein, pomc proopiomelanocortin, cart cocaine amphetamine - related transcript, amp adenosine monophosphate, ampk amp kinase, acc acetyl - coa carboxylase . Black arrows activation; discontinued red lines inhibition; thin discontinued red line weak activation [4, 51, 52, 54, 57, 58, 72, 182]fig . Luminal fructose induces weak release of 5-ht and glp-1 from enteroendocrine and l cells, respectively, weak pyy, insulin and leptin secretion, as well as weak ghrelin suppression . Many neuronal signals are communicated via the vagus nerve to the brain stem, which relays the glucose signal to hypothalamic nuclei and then to the pertinent target cells: npy / agrp and pomc / cart neurons . Npy neuropeptide y, agrp agouti - related protein, pomc proopiomelanocortin, cart cocaine amphetamine - related transcript, amp adenosine monophosphate, ampk amp kinase, acc acetyl - coa carboxylase . Black arrows activation; discontinued red lines inhibition; grey arrows low secretion or low activation [4, 37, 51, 52, 54, 58, 59, 72, 182] hypothetical model of the peripheral and central effects of glucose on food intake . Vagal afferents express glp-1 and 5-ht receptors, and are implicated in the regulation of insulin secretion . Many neuronal signals are communicated via the vagus nerve to the brain stem, which relays the glucose signal to hypothalamic nuclei and then to the pertinent target cells: npy / agrp and pomc / cart neurons . Npy neuropeptide y, agrp agouti - related protein, pomc proopiomelanocortin, cart cocaine amphetamine - related transcript, amp adenosine monophosphate, ampk amp kinase, acc acetyl - coa carboxylase . Black arrows activation; discontinued red lines inhibition; thin discontinued red line weak activation [4, 51, 52, 54, 57, 58, 72, 182] hypothetical model of the peripheral and central effects of fructose on food intake . Luminal fructose induces weak release of 5-ht and glp-1 from enteroendocrine and l cells, respectively, weak pyy, insulin and leptin secretion, as well as weak ghrelin suppression . Many neuronal signals are communicated via the vagus nerve to the brain stem, which relays the glucose signal to hypothalamic nuclei and then to the pertinent target cells: npy / agrp and pomc / cart neurons . Npy neuropeptide y, agrp agouti - related protein, pomc proopiomelanocortin, cart cocaine amphetamine - related transcript, amp adenosine monophosphate, ampk amp kinase, acc acetyl - coa carboxylase . Black arrows activation; discontinued red lines inhibition; grey arrows low secretion or low activation [4, 37, 51, 52, 54, 58, 59, 72, 182] the mesolimbic dopamine (da) system plays a critical role in the reinforcing effects of reward and is implicated in conditions such as drug addiction and eating disorders . Dopamine is the primary neurotransmitter involved in the brain reward pathways and in the reward value of sweet taste, mainly because sweet taste activates mesolimbic da circuits involved in the mediation of natural as well as drug rewards . Mice lacking the cellular machinery required for sweet taste transduction (trpm5) learned to prefer the postingestive effects of sucrose . These mice did not develop a preference for sucrose per se probably due to the short training sessions . However, zuckerman et al . Reported that trpm5 ko mice learned to prefer glucose but not fructose solutions in 24-h two - bottle choice tests . Furthermore, trpm5 ko mice developed a robust preference for sucrose solutions based solely on caloric content . Sucrose intake induced da release and increased neuronal responses in the ventral striatum of these mice in the absence of gustatory input . These findings suggested that calorie - rich nutrients could directly influence brain reward circuits that control food intake independently of palatability or functional taste transduction . Dopamine release is stimulated in the nac by the sweet taste in the mouth . It was reported that the neurochemical effects observed with intermittent sugar access are not only due to sucrose postingestive properties but also to the sweet taste of sucrose . The mesolimbic da projection from the ventral tegmental area (vta) to the nac data from animal models of binge - eating but with normal weight show that behavioural and neuronal consequences of bingeing on a palatable food are different from those that result from simply consuming the palatable food in a non - binge manner, i.e. Physiological, neural, and behavioural effects independent of a dio . Excessive intake of palatable foods under specific conditions can produce behaviours and changes in the brain that resemble an addiction - like state, such as greater activation of the anterior cingulate cortex, the medial orbitofrontal cortex, and the amygdala, regions associated with motivation . These changes may be more closely correlated with binge - eating behaviour than they are to body weight . It was recently suggested that people at risk of obesity initially show a hyperfunctioning in the gustatory and somatosensory cortices that increases pleasure perception from food, leading to increased reward, overeating, and weight gain . This overeating may induce receptor down - regulation in the striatum, increasing the likelihood of further overeating and continued weight gain . Obese versus lean humans show less activation in the dorsolateral prefrontal cortex, and greater activation of regions involved in the reward value of stimuli (striatum, amygdala, orbitofrontal cortex, and mid - insula), in attention regions (ventral lateral prefrontal cortex), and in somatosensory regions, in response to high - fat / high - sugar food images relative to control images . Similarly, dio minipigs showed deactivations in the dorsolateral and anterior prefrontal cortices and activations in the ventral posterior nucleus of the thalamus and middle temporal gyrus, compared to lean minipigs . Moreover, the anterior and dorsolateral prefrontal cortices as well as the insular cortex activity were negatively associated with body weight . These data suggested that the reduced activation of the prefrontal cortex observed in obese subjects is an acquired feature of obesity . Mccutcheon et al . Showed that da release in the nac core to food - predictive cues is strongly modulated by food s characteristics . Cues paired with sucrose (the preferred reward) evoked greater da release, than cues predicting saccharin (the non - preferred reward). This subjective preference and greater da release could result from a difference in the orosensory qualities of the pellets or an aversion to the bitter taste of saccharin, and suggests that sucrose may more powerfully motivate behaviour . The nac, the amygdala, and the medial prefrontal cortex are implicated as central sites of action for the suppressive effects of the da-1 receptor (d1r) and da-2 receptor (d2r) antagonists (sch23390 and raclopride, respectively), on sugar intake and on the expression of flavour preferences conditioned by the sweet taste of sugars . Subcutaneous administration of d1r agonist prior to food preference test reduced the intake of the regular diet and induced a strong preference for high - fat / high - sucrose food, whereas d2/d3 receptor agonist had the opposite effects . These results suggested that the da receptor subtype is a major determining factor of the direction in which sweet food preference is modulated, in addition to the level of da release in the nac . Subcutaneous injections of d1r and d2r antagonists substantially and dose dependently reduced the intake and preference of sucrose solutions but not saccharin solutions, suggesting that the antagonists decreased the reinforcing value of sucrose, but not saccharin, solution . Systemic administration of dopamine d1r (sch23390) and d2r (raclopride) antagonists in the medial prefrontal cortex and amygdala, but not in the nac shell, blocked the acquisition and expression of fructose - conditioned flavour preferences in rats . In contrast to the effect of intragastric glucose following bilateral injection of the d1r antagonist on blocking the acquisition of conditioned flavour preferences, bilateral hypothalamic injections of d1r and d2r antagonists failed to alter the acquisition of fructose - conditioned flavour preference in rats . These findings suggest an important difference between flavour flavour and flavour nutrient - conditioned flavour preferences . Fructose intake and fructose - conditioned flavour preference appear to be more dependent on d2r activity than sucrose intake . Injections of d1r antagonist into the amygdala or nac during training did not block fructose conditioning, but did block the acquisition of a flavour preference produced by glucose infusions . Rats under a high - fat diet responded to all raclopride doses with reductions in sucrose intake, but not in fructose intake, while rats fed a high - fat and sugar diet only responded to the highest dose of raclopride, with significant reductions in fructose intake . These data indicate that there is a differential involvement of d1r and d2r in flavour flavour and flavour nutrient preference, respectively . An explanation may include differential neural and hormonal postingestive effects exerted by fructose and sucrose . In this context, it may be possible that the high - fat diet and the high - fat and high - sugar diet altered sucrose and fructose preferences differently as a result of their differential effects on oral and gastrointestinal signals upstream the reward system . Anatomical and functional evidence demonstrates multiple interactions between the mesolimbic da system and peripheral signals regulating food intake . One such system is the cck system . In an obese rat model lacking the cck-1 receptors (oletf), treatment with d2r antagonist raclopride, but not d1r antagonist sch23390, showed increased potency to reduce sucrose real intake, indicating that d2r are involved in heightened increased consumption of sucrose observed in these obese rats . These findings confirm the notion that da increases sucrose intake due to the assignment of an actual rewarding value of sucrose primarily based on its sensory stimulatory effects . They also confirm that altered da signalling present in obesity is involved in the increased potency of sucrose palatability to maintain ingestion in obese subjects . Ghrelin has recently emerged as a potent modulator of the mesolimbic dopaminergic reward pathway, suggesting a role for ghrelin in food reward . Ghrelin targets a key mesolimbic circuit involved in food and drug - induced reinforcement, i.e. Da projection from the vta to the nac . Skibicka et al . Identified the vta, a key structure in the mesolimbic reward system, as a primary target for ghrelin s effects to increase motivation for a sweet food reward . Conversely, blockade of ghs - r1a (ghrelin type 1a receptor) signalling significantly decreased operant responding for sucrose . These findings indicate that ghrelin plays an important role in motivation and reinforcement for sucrose . They suggest that ghrelin antagonists have therapeutic potential for the treatment for obesity and for suppressing sweet food overconsumption [143, 144]. Lustig reviewed that chronic hyperinsulinemia may prevent da clearance from the nac and leptin signalling, leading to leptin resistance and increased food intake . Thus, by promoting insulin resistance and hyperinsulinemia, fructose excessive intake may alter da neurotransmission and the hedonic response to food leading to overeating . Increasing the palatability of food by the addition of sucrose undermines normal satiety signals and motivates energy intake independent of energy needs . Animal models of intermittent sugar administration can induce behavioural alterations consistent with dependence, i.e. Bingeing, withdrawal and anxiety, craving, and cross - sensitisation to other drugs of abuse . Anatomical and functional evidence demonstrates multiple interactions between the mesolimbic da system and peripheral signals regulating food intake . One such system is the cck system . In an obese rat model lacking the cck-1 receptors (oletf), treatment with d2r antagonist raclopride, but not d1r antagonist sch23390, showed increased potency to reduce sucrose real intake, indicating that d2r are involved in heightened increased consumption of sucrose observed in these obese rats . These findings confirm the notion that da increases sucrose intake due to the assignment of an actual rewarding value of sucrose primarily based on its sensory stimulatory effects . They also confirm that altered da signalling present in obesity is involved in the increased potency of sucrose palatability to maintain ingestion in obese subjects . Ghrelin has recently emerged as a potent modulator of the mesolimbic dopaminergic reward pathway, suggesting a role for ghrelin in food reward . Ghrelin targets a key mesolimbic circuit involved in food and drug - induced reinforcement, i.e. Da projection from the vta to the nac . Identified the vta, a key structure in the mesolimbic reward system, as a primary target for ghrelin s effects to increase motivation for a sweet food reward . Conversely, blockade of ghs - r1a (ghrelin type 1a receptor) signalling significantly decreased operant responding for sucrose . These findings indicate that ghrelin plays an important role in motivation and reinforcement for sucrose . They suggest that ghrelin antagonists have therapeutic potential for the treatment for obesity and for suppressing sweet food overconsumption [143, 144]. Lustig reviewed that chronic hyperinsulinemia may prevent da clearance from the nac and leptin signalling, leading to leptin resistance and increased food intake . Thus, by promoting insulin resistance and hyperinsulinemia, fructose excessive intake may alter da neurotransmission and the hedonic response to food leading to overeating . Increasing the palatability of food by the addition of sucrose undermines normal satiety signals and motivates energy intake independent of energy needs . Animal models of intermittent sugar administration can induce behavioural alterations consistent with dependence, i.e. Bingeing, withdrawal and anxiety, craving, and cross - sensitisation to other drugs of abuse . The endocannabinoid system is a lipid - signalling system composed of three non - ubiquitous receptors (cb1, cb2, and likely cb3 receptor), two endogenous ligands (i.e. Anandamide and 2-arachidonoyl - glycerol, 2-ag), and the enzymatic machinery for their synthesis and degradation . This system is implicated in the regulation of appetite, eating behaviour, and body weight homeostasis at both peripheral and central levels . In the brain, the endocannabinoid system appears to control food intake mainly at three functional levels, i.e. The hypothalamus, the dorsal vagal complex, and the limbic system, by affecting satiety signals and interacting with brain reward pathways [148, 149]. Previous studies have revealed the ability of marijuana, or of its main psychoactive component -tetrahydrocannabinoid (-thc), to induce not only hyperphagia, but also to increase the desire to consume highly palatable food and to impact food selection concomitantly . Endocannabinoids have been implicated in the regulation of consumption of palatable food, sugar in particular . Cb1 receptor antagonist (sr 141716, also known as rimonabant, an anorectic anti - obesity drug) resulted in reduced body weight and appetite for sweet foods and drinks . Following low oral doses of -thc (0.25 and 0.40 mg / kg), there was a dose - dependent increase in preference for palatable food and sucrose intake in rats . Similarly, administration of -thc (0.5, 1.0, and 3.0 mg / kg), anandamide (1.0 and 3.0 mg / kg), and 2-ag (0.2, 1.0, and 2.0 mg / kg) substantially increased the number of licks of 10% sucrose solution, due to increased bout duration rather than bout number, whereas administration of cb1 antagonist sr141716 significantly decreased total licks . Rimonabant also specifically reduced sucrose, alcohol, and sweet food intake in rats and marmosets . Endocannabinoids (anandamide and 2-ag) peripherally administered selectively enhanced gustatory nerve (chorda tympani) responses and electrophysiological responses of taste cells, located on the anterior tongue innervated by the chorda tympani nerve, to sweeteners (i.e. Saccharin, glucose, and sucrose) in mice . These sweet - enhancing effects of endocannabinoids were mediated by cb1 receptors, which were coexpressed in taste cells with the sweet receptor subunit t1r3 in taste cells . However, since no differences were previously found in gustatory nerve (chorda tympani) responses to glucose, sucrose, and fructose in pigs, one might speculate that endocannabinoids may exert similar degree of taste enhancement sensitivities between these three sugars . But this hypothesis remains unclear since neural responses do not always predict functional sensitivity; moreover, the observed responses do differ at various stimulus concentrations, and it is unclear how endocannabinoids would interact with this . Intraperitoneal administration of endocannabinoids to wild - type mice selectively enhanced gustatory nerve responses and electrophysiological responses of taste cells, located on the anterior tongue innervated by the chorda tympani nerve, to sweet compounds (sucrose, saccharin, and glucose). These sweet - enhancing effects of endocannabinoids were mediated by cb1 receptors, which are coexpressed in taste cells with the sweet receptor component t1r3 . Consumption of fructose solution in combination with standard rat chow resulted in increased mrna levels of cb1 in the rat hypothalamus . Intake of sucrose, glucose, and fructose solutions during 7 days affected the mrna expression of the majority of enzymes involved in the synthesis and degradation of anandamide and 2-ag, in rats . Fructose solution increased mrna levels of fatty acid amide hydrolase (faah) (involved in anandamide degradation), compared to water, glucose, and sucrose solutions . This suggests that fructose intake might induce an overproduction of anandamide and that an up - regulation of this enzyme is necessary to maintain normal levels of anandamide . The three sugar solutions induced a down - regulation of phospholipase c (involved in anandamide synthesis). This may suggest an attempt to maintain anandamide at physiological levels during periods of high - sugar consumption irrespective of the nature of the sugar . Monoglyceride lipase (mgll), the main enzyme involved in 2-ag degradation, was also down - regulated by the three sugar solutions compared to water intake . This would suggest that 2-ag is degraded less readily in rats drinking sugar solutions than in water drinking rats . However, only fructose intake increased mrna levels of diacylglycerol lipase 1 (involved in 2-ag synthesis), suggesting that more 2-ag is being produced . However, the interpretation of these results is conflicting, given the simultaneous increase or decrease in enzymes involved in the synthesis and degradation of endocannabinnoids, respectively, induced by the sugar solutions . Besides, neither protein levels nor actual concentrations of endocannabinoids were measured, making it difficult to draw a conclusion . . Demonstrated that a diet - induced obesity (dio) by excess dietary lipid intake is associated with altered expression of cb1 mrna, higher plasma endocannabinoids, or increased adipose tissue endocannabinoid synthesis . Blockade of cb1 receptor improves the gut barrier and reduces metabolic endotoxemia, by a mechanism independent of eating behaviour, suggesting a control of gut permeability by cb1 receptors through interactions with gut microbiota . It is possible that fructose could also modulate the intestinal endocannabinoid system by a similar mechanism, but future studies are needed to investigate this hypothesis . Finally, endocannabinoids have been proven to interact with brain reward pathways in a manner similar to other reward - enhancing drugs . Therefore, the endocannabinoid system might affect eating behaviour through the modulation of the reward circuit . Which are the effects of the different dietary sugars on the endocannabinoid system and their interaction with brain reward pathways to affect eating behaviour as presented in the previous section on the da system, different types of sugars differentially modulate this system . Therefore, it is possible that these differences might also be observed in the endocannabinoid system and this requires further investigation . . Chronic suppression of the endogenous -opioid receptor signalling in the nucleus accumbens (nac) shell and core significantly attenuates the development of a dio by reducing the intake of palatable, high - sugar foods in rats . Opioid antagonism in the nac is associated with a reduction in sweet food preference and sucrose intake, and weakens hedonic properties of sucrose and motivation for sucrose [156158]. Conversely, stimulation of -opioid signalling in the nac increases sucrose intake and motivation . The consumption of sweet tastants results in neurochemical changes within the brain, which may reflect a shift in opioid - mediated responses . Sucrose and glucose intake paired with opioid receptor antagonism (naloxone) induced an increase in the number of c - fos - positive nuclear profiles and an elevation in opioid -1 receptor binding in the cingulate cortex, hippocampus, locus coeruleus, and accumbens shell, associated with the presence of opiate withdrawal - like symptoms, such as teeth chattering . These results suggest that ingestion of sucrose and glucose induces neurochemical changes within the opioid brain circuitry . Opioids support a drive to consume sugar, and this mechanism is mainly dependent on their ability to act through the reward system . Similar to the reports in the cocaine studies for drug euphoria and craving, sweet liking may increase with the dose while sweet wanting may not . Overall, opioid signalling, particularly through its -receptor in the nac, is involved in the expression of reward behaviours induced by the consumption of sweet - palatable foods and may be involved in the development of dio . However, the vast majority of these studies, in both animals and humans, regarding opioids and sugar intake have used sucrose as the source of sugar . One of the few studies using both glucose and fructose was performed by bernal et al . . They found that rats develop strong preferences for flavours paired with the sweet taste of fructose or the post - oral nutrient effects of glucose . Opioid antagonism at the nac shell and core did not block sugar - conditioned flavour preference at any dose and with neither glucose nor fructose solutions . However, the authors did not directly compared to glucose versus fructose stimulus so there is no clear evidence about possible differences between these sugars following opioid antagonism in the nac . Given the high amounts of fructose currently consumed in western diets, it would be interesting to directly compare the effects of free fructose and glucose to those observed with sucrose on the opioid system . Given the observations of the effects of fructose intake on appetite, its higher palatability compared to glucose, and its differential effects at both peripheral and central levels, including the da system, one might speculate that fructose could induce more profound effects on this system than sucrose or glucose . However, since fructose may not cross the bbb at typical intake levels, this hypothesis seems unrealistic . Further research directly comparing the effects of sucrose, glucose, and fructose is needed to investigate this hypothesis . Several pieces of evidence have led to the assumption that fructose excessive intake may be responsible for the increasing prevalence of obesity since the last decades . This has stimulated research aiming at understanding the underlying mechanisms of this fructose - induced obesity . For example, epidemiologic and experimental evidence indicates that a greater consumption of sugar - sweetened beverages with hfcs is, in fact, associated with weight gain and obesity, and that hfcs accounts for 40% of caloric sweeteners used in the united states [13, 16, 17, 164, 165]. In addition to these epidemiological data, evidence has shown that fructose induces smaller increases in insulin, leptin, and other satiety peptides compared to glucose [37, 5559]. This suggested an endocrine mechanism by which fructose might induce greater food intake and weight gain than glucose . However, while it is true that fructose intake in the form of hfcs makes up a significant proportion of energy intake in the western diet [13, 164], it is also true that this increase in fructose intake is necessarily associated with an increase in total energy intake and in glucose (from hfcs). Besides, equal amounts of glucose and fructose are necessary for maximal fructose absorption in humans . This makes questionable the effect of fructose per se for increasing food intake, inducing weight gain and metabolic diseases . In fact, there are some well - controlled studies showing divergent findings in this regard that are important to discuss here given the extended great concern regarding the fructose - induced obesity . . Found no differences in terms of food intake, pyy, and leptin serum levels following 2-week intake of sucrose, glucose, or fructose solutions in rats . Moreover, these authors found that the fructose - drinking group had the smallest increase in food intake, probably attributed to the lowest intake of fructose solution . This was attributed to the fact that fructose is sweeter than sucrose and glucose, and it may also be attributable to the short period (2 weeks) of sugar solution intake . In addition, no difference was found in energy intake and weight gain following 50-day intake of sucrose, glucose, or fructose solutions, but body adiposity increase was greater with sucrose than with fructose solutions . In humans, no differences in terms of energy intake, satiety, and energy compensation, nor in plasma glucose, glp-1, insulin, and ghrelin release were found following acute ingestion of preload drinks containing sucrose or hfcs (1.5 mj). This lack of difference in satiety was found despite different biochemical properties (leading to different transport across the gut epithelium and thus different transit time) as well as different mechanisms underlying satiety between sucrose- and hfcs - containing drinks [64, 110, 168]. Therefore, more studies are needed to clarify these discrepancies . Glendinning et al . Investigated in four strains of mice given free access to sugar solutions and showed that sucrose promoted more overeating, resulting in increased weight gain and adiposity compared to fructose, regardless of mouse strain . These authors reported as well that mice and rats consume less fructose than isocaloric sucrose . Fructose orally ingested or intraduodenally infused induced insulin release and inhibited food intake more than glucose in rats and humans [7, 170, 171], whereas another study found no difference between these sugars orally ingested on food intake . Sclafani and ackroff reported that 16% glucose intragastric infusion condition a strong flavour preference in mice, whereas fructose and galactose infusions failed to do so . The latter findings are opposed to other findings, suggesting that fructose is a weaker elicitor of satiation signals [54, 62] and may have a more rewarding effect probably due to its higher sweetness than glucose . The only rodent study that appears in the literature reporting fructose - induced weight gain more than sucrose - fed mice used a 15% fructose solution or a 10% sucrose solution, which invalidates the findings . White recently reviewed that many animal studies have used extremely high - fructose doses, or altered the usual glucose - to - fructose ratio that are not predictive of typical human diets, leading to abnormal metabolism . He exposed as well that (i) the increased energy intake per capita coupled with insufficient compensating exercise is a more consistent explanation to the obesity epidemics; (ii) there has been no positive correlation between fructose intake and increasing rates of obesity; (iii) consumption of added sugars has not increased, but actually decreased for more than a decade; and (iv) all sources of fructose in human diets contain comparable amounts of glucose, and glucose is the dominant sugar in the human diet (5 times more glucose than fructose). The fructose hypothesis is refuted by studies using real - world fructose exposures showing no differential effects versus control, and cause - and - effect evidence of adverse effects is lacking at typical human exposure levels and patterns . Sun et al . Analysed the intake patterns of> 25,000 subjects in the nhanes 19992006 databases and found that daily fructose intakes with the american diet averaged 9% of daily intake, that fructose is rarely consumed solely or in excess over non - fructose sugars, and that fructose and non - fructose sugar ordinary intake was not positively associated with indicators of metabolic syndrome, uric acid, or bmi . A metaanalysis by sievenpiper et al . Reported the effects of fructose on body weight in controlled feeding trials . They found that fructose has no effect on body weight in isocaloric trials (637 participants) compared to isocaloric diets containing a non - fructose sugar . In contrast, high doses of fructose in hypercaloric trials (119 participants) induced weight gain . The effect of fructose - induced body weight gain in hypercaloric trials may have been due to excess energy intake rather than fructose itself because (i) weight gain is similar to that which would be predicted with consumption of a 2,000-kcal diet with similar amount excess energy; (ii) high - precision estimates of energy expenditure, fat, and carbohydrate oxidation using whole - body calorimetry showed no differences among fructose, glucose, or sucrose . Taken together, these data suggest that an excess energy may be a more important factor for weight gain than the type of sugar . Concerning some of the central effects, both oral intake of glucose and fructose solutions during 2 weeks produced a down - regulation of pomc mrna levels . Processing of pomc by pro - hormone convertases results in the production of -msh, which suppresses feeding, and - endorphin, which stimulates it . Thus, pomc mrna decrease, together with a decrease in - endorphin, may indicate the down - regulation of a potent suppressor of food intake and less rewarding signals through the opioid pathway by sugar solutions . Thus, the observed reduction in npy mrna levels in this study may attempt to balance for calorie overconsumption . Despite the down - regulation of hypothalamic npy and pomc mrna, there was no reduction in hyperphagia induced by the consumption of sugar solutions . Taken together, fructose hypothesis remains controversial, and a cause - and - effect association between fructose intake and the metabolic syndrome and obesity has not been clearly confirmed yet . Thus, there is a need for more research on fructose with experimental designs based on physiological conditions so that a consensus could be established . The aim of this review was to critically discuss the effects of dietary sugars at both central and peripheral levels . Based on the current findings, diverse hypotheses were postulated all along the review sections with two main goals: (i) to open new perspectives for future research that may contribute to our understanding of current data with a special focus on fructose, and (ii) to clarify controversial findings in order to advance in the establishment of a consensus concerning the differential effects of the main dietary sugars found in humans diets at both peripheral and central levels . In summary, contrary to glucose, excessive fructose intake may provoke metabolic disturbances, such as an increased gut permeability, low - grade inflammation, nafld, insulin resistance, and dyslipidaemia . Through luminal gut detection and following the activation of sweet taste receptors, glucose triggers the secretion of peripheral anorexigenic peptides, i.e. Insulin, leptin, glp-1, pyy, and suppression of orexigenic peptides (e.g. Ghrelin) that activate vagal pathways and act on brain target regions controlling appetite (e.g. The arcuate nucleus of the hypothalamus and the dorsal vagal complex), thus leading to appetite suppression and reward response . Glucose may also directly induce its effects on appetite suppression by crossing the bbb, where it suppresses ampk activity, an effect that stimulates neuronal activity of pomc / cart expressing neurons, which contributes as well in the satiety response . Fructose may have different effects on the secretory profile of appetite peptides and neuropeptides, leading to reduced appetite suppression as well as an indirect effect on the reward response, i.e. Through a deficient stimulation of leptin and insulin secretion, hormones implicated in the rewarding effects of palatable food, fructose may provoke a deficient reward response leading to overeating . However, most of these results were obtained from rodents and using extremely high doses of fructose far from the typical human diets . Other studies found small or no differences between glucose, sucrose, and fructose in appetite peptide secretion, food intake, and weight gain . These discrepancies may be due to differences in species, metabolic phenotype, experimental approaches, form of administration (peripheral or central infusions), in the form of solutions or added in the diet, doses, duration of exposure, and experimental diet compositions . Several clues and hypothesis were proposed for future research aiming at clarifying these controversial findings . Besides, fructose is rarely consumed isolated in the diet, but rather in the form of hfcs or sucrose, or consumed along with glucose . Therefore, no definite conclusions could be established for giving any nutritional recommendations to suppress or reduce fructose from the human diets . Overall, data obtained from well - controlled studies in pigs or rodents, and epidemiological studies in humans, suggest that it is the association between fructose and other components in the diet, such as fat, cholesterol, and other dietary sugars, as well as total caloric intake coming from dietary sugars, responsible for the metabolic effects and weight gain, rather than fructose intake per se . Given the difficulty to perform controlled studies in humans for ethical reasons, a valuable approach may be through the use of the pig model that has been shown to present greater similarities to humans than smaller animals (e.g. Cats and rodents). At the central level, consistent evidence has suggested the capacity of fructose to induce changes in neuropeptides or brain activity, with a resulting decrease in the satiety response . However, there is no clear evidence of the capacity of fructose orally ingested to cross the bbb . Thus, it seems unlikely that fructose could directly induce changes in brain appetite peptides to produce its effects on satiety . Fructose is partially (12%) metabolised in the gut during absorption, and the liver and kidneys rapidly metabolise the remaining fraction . This leads to very low fructose plasma concentrations and during a very short time, as well as the low glut5 affinity for fructose and low glut5 concentrations in the bbb, and the possibility of a fructose malabsorption when ingested in high doses . These factors make unlikely that fructose could cross the bbb and induce significant effects on the brain . Contrary to the well - established direct central effects of glucose on energy homeostasis and food intake through central glucose - sensing mechanisms, the fructose effects on eating behaviour are more likely to be exclusively through indirect mechanisms, i.e. Via activation of t1r2/t1r3-sensing mechanism in the mouth and gut, as well as intestinal glucose transporters . This activation triggers the secretion of appetite peptides that may affect thereafter brain neuropeptides involved in appetite control, as well as the activation or deactivation of brain regions involved in appetite and reward . Therefore, fructose may indirectly influence appetite and reward through changes in the levels of peripheral neural input or metabolic intermediaries modifying the activation of brain regions implicated in appetite and reward . While fructose effects on the reward circuitry seem to be consistent when administered i.c.v . Or intragastrically, this review presented consistent evidence showing the implication of the endocannabinoid, opioid, and mesolimbic dopaminergic systems in the modulation of sweet taste reward and in the development of preferences for sweet taste that may lead to aberrant eating behaviours . This addictive - like condition could be explained by a desensitisation of the reward pathways . However, most of these studies used sucrose as the source of sugar in the experiments, thus making it impossible to separate the specific effects of glucose and fructose or their interaction . The apparent parallel increases between fructose intake and obesity development make necessary more research to elucidate possible differences between glucose, sucrose, and fructose on the reward circuitry . For example, the satiety hormones insulin and leptin are implicated in the reward effect from palatable food . In this regard, a hypothesis was postulated arguing that if fructose induces lower leptin and insulin secretions, it may also induce less satiety effect and a less rewarding effect compared to glucose intake, and therefore an increase in food intake to compensate for this lack of reward from food . However, the opposite may also be plausible: if fructose is less rewarding than glucose, it may not stimulate excessive intake, as is the case of several studies reported in the controversial findings section . This may be another angle for weighing detrimental effects of sugars and thus should be investigated in future studies . We presented results showing that sucrose intake induces da release and increases neuronal activity in the brain reward circuitry . Dopamine release is stimulated in the nac by the sweet taste in the mouth and by the postingestive actions of sugars . Considering that fructose is sweeter than glucose and sucrose for humans, one might hypothesise that fructose could induce greater da release compared to glucose and sucrose, which may lead to increased reward from food, increased food intake, and aberrant eating behaviours . However, this hypothesis remains controversial since calorie - rich nutrients (i.e. Sucrose) can directly influence brain reward circuits that control food intake independently of palatability or functional sweet taste transduction . On the other hand, most of the studies comparing the effects of intermittent and/or excessive sugar intake with the effects of addiction to drugs on the dopamine system have used sucrose as well, and very few have used glucose; to our knowledge, none has compared fructose versus glucose on the characteristics of addiction (e.g. Escalation of intake). It was until recent years that researchers began to compare the effects of da receptor antagonists on the expression of fructose - conditioned preferences compared with sucrose . These studies have mainly found that fructose intake and fructose - conditioned flavour preference appear to be more dependent on d2r activity than sucrose intake (e.g. [136, 138, 139]). Therefore, future studies should directly compare da release levels, da transporter expression and da receptor expression patterns following ingestion of glucose, sucrose, and fructose . Concomitantly, these studies should also measure appetite peptide secretion levels, neuropeptides, brain activity of regions implicated in appetite and reward, and feeding behaviour tests (e.g. Food choice, eating microstructure, operant conditioning, and progressive ratio). This integrated approach may clarify the possible links between satiety and reward effects induced by the ingestion of different dietary sugars . While some recent evidence exists showing a differential effect between glucose and fructose on the function of brain regions implicated in appetite and food reward, this concept needs to be confirmed in humans and a non - rodent animal, under controlled experimental conditions and using a physiological fructose intake . An additional question that should be addressed in future studies is which are the underlying mechanisms leading to fructose effects on brain functions, considering that this sugar might not be able to cross the bbb and directly produce these observed effects . In this regard, several hypotheses were presented here that may contribute to address this question in future studies . Future research should therefore focus on resolving the apparently inconsistent findings, suggesting that excessive fructose intake may promote adverse effects at both peripheral and central levels to a greater extent than those provoked by glucose or sucrose . There is a particular need to integrate the metabolic, behavioural, and neurological effects of these sugars . An approach combining behavioural (e.g. Progressive ratio) and metabolic (e.g. Plasma and protein levels of peptides and neuropeptides) measurements, pet, and fmri imaging, together with the use of an animal model closer to humans (i.e. The pig), would contribute to an improved understanding of the complexity of the development of diseases induced by dietary sugars.
Dengue virus (denv) causes most of the world s mosquito - borne viral infections, and is the etiologic agent of dengue fever (df), dengue hemorrhagic fever (dhf), and dengue shock syndrome (dss). One of the first lines of host immunity against denv is the type i interferon (ifn - i or ifn/) response, which inhibits viral replication, and sets the stage for the development of adaptive immunity . Denv - mediated degradation of signal transducer and activator of transcription 2 (stat2), a component of the ifn/ signaling pathway, has emerged as an important determinant of denv pathogenesis and host tropism . Here we review the strategies that denv uses to evade the type i interferon response, and postulate how studying denv ns5-mediated stat2 degradation may contribute to the development of immunocompetent denv mouse models and anti - denv therapeutics . There are over 50 million denv infections and approximately 500 000 cases of dhf / dss annually . More than 2.5 billion people live in the warm climes that are home to expanding populations of aedes aegypti and aedes albopictus mosquitoes, the vectors of denv . Many denv infections are asymptomatic or show only mild symptoms but df and dhf / dss occur in a subset of patients . Older names for df include coup de barre (beating with a stick) and break bone fever to describe the intense headache, myalgia and bone pain that accompany the disease . Df progresses to dhf / dss when patients develop capillary leakage, thrombocytopenia and liver damage . Because host immunity is an important contributor to denv pathogenesis, focusing on the interaction between denv and the host immune response is a promising approach to the development of drugs and vaccines against denv . The recent discovery of denv-5 brings the total number of known denv serotypes to five . The flavivirus genus includes important arthropod - borne viruses such as west nile virus (wnv) and japanese encephalitis virus (jev). In addition to its role as the viral genetic material, the genome functions as an mrna whose translation yields a polyprotein that is cleaved by host proteases and the viral ns2b/3 protease to give the structural and nonstructural proteins of the virus (fig . 1). There are three structural proteins, capsid (c), premembrane / membrane (prm / m), and envelope, and seven nonstructural proteins, ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5 (fig . The nonstructural proteins mediate replication of the viral rna and antagonism of the host immune response, while the structural proteins encapsulate newly copied viral rnas into denv virions . Figure 1 . The denv virion and genome . (a) denv contains a capped plus - strand rna genome that is surrounded by a shell composed of capsid (c) proteins . The capsid is enveloped by a lipid bilayer embedded with envelope (e) and membrane (m) proteins that mediate virus entry into susceptible cells . (b) the denv genome functions as an mrna whose translation yields a polyprotein that is processed by the viral ns2b/3 protease and host proteases to give the structural and nonstructural proteins of the virus . Denv replicates in a variety of human cell types including endothelial cells, fibroblasts, dendritic cells (dcs), macrophages, and b cells . Infection of these cells leads to activation of the type i interferon (ifn - i or ifn/) response, an innate immune mechanism that protects the host against viral infections . During viral replication, double - stranded rna and other pathogen - associated molecular patterns (pamps) accumulate within the cell . The recognition of pamps by pattern recognition receptors such as retinoic - inducible gene i (rig - i), melanoma differentiation - associated protein 5 (mda-5), and toll - like receptor 3 (tlr3) leads to signaling events that culminate in ifn/ production . In vivo and cell culture experiments have shown that tlr3, rig - i, and mda-5 contribute to ifn production in response to denv infection but that rig - i and mda-5 may serve redundant roles . Denv - infected cells produce far less ifn/ than cells infected with more potent inducers of ifn/ such as sendai virus due to the cleavage of sting, an adaptor protein that is believed to function downstream of mda-5 and rig - i as well as cyclic guanosine monophosphate - adenosine monophosphate synthase (cgas), a sensor of cytoplasmic dsdna . Despite this immune evasion mechanism, ifn production is not completely halted by ns2b/3 protease, and ifn/ is secreted from infected cells as denv infection proceeds . Ifn/ signaling ensues when ifn/ from an infected cell binds to type i ifn receptors (ifnar1/2) found on the surface of the infected cell or nearby cells . Ifnar1/2 engagement leads to the activation of janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2), two tyrosine kinases that physically associate with ifnar1/2 . Tyk2 and jak1 phosphorylate signal transducer and activator of transcription 1 (stat1) and signal transducer and activator of transcription 2 (stat2), which interact with interferon regulatory factor 9 (irf9) to form ifn - stimulated gene factor 3 (isgf3), a complex that recognizes ifn - stimulated response elements (isres). Binding of isgf3 to the isres of ifn - stimulated genes (isgs) leads to the transcription of isgs . For example, interferon - induced transmembrane proteins 1, 2, and 3 (ifitm1, ifitm2, and ifitm3) inhibit early steps in dengue replication while viperin, interferon - stimulated gene 20 (isg20), and dsrna - activated kinase (pkr) inhibit the synthesis of denv macromolecules . Previously uncharacterized isgs such as ifn-inducible protein 6 (ifi6), hepanarase (hpse), and n - ethylmaleimide - sensitive factor attachment protein (napa) have also been identified as inhibitors of denv replication . In fact, denv has devoted a significant portion of its genome to antagonizing human type i ifn signaling . Ns2a, ns4a, and ns4b have been shown to inhibit stat1 phosphorylation while ns5 has been shown to mediate proteasome - dependent stat2 degradation . However, these four viral proteins also have roles that are distinct from ifn/ signaling antagonism . For example, ns2a is required for virion assembly, while ns4a and ns4b are required for induction of the membranes upon which viral replication occurs, and for organization of the replication complex, respectively . Expression of any of the three decreases isre promoter activation and stat1 phosphorylation, but their combined expression has an even stronger inhibitory effect . The ns5 n - terminus encodes a methyl transferase that induces methylation of guanine n7 and 2-hydroxyl ribose of the viral rna cap . These modifications are required for viral replication and for evading the antiviral ifn - induced tetratricopeptide repeat (ifit) proteins, respectively . The n - terminus of ns5 also contains a guanylyltransferase, which is required for 5 rna cap synthesis . S most studied role is that of the viral rna - dependent rna polymerase (rdrp), which is encoded by its c - terminal domain . Ns5 mutants that are deficient in one or more roles while being proficient in others have been characterized, and it is thought that phosphorylation may serve as a switch among the various functions . Over the past decade, flavivirus ns5 proteins have surfaced as potent antagonists of ifn signaling . However, ns5 proteins of different flaviviruses accomplish their ifn signaling inhibition in disparate ways . For example, wnv and jev inhibit ifn signaling by preventing phosphorylation of signaling proteins while denv ns5 promotes the proteasomal degradation of human stat2 . Degradation of stat proteins is a common mechanism of virus - mediated ifn signaling inhibition . For instance, expression of the v proteins of human parainfluenza virus 2 (hpiv2) or parainfluenza virus 5 (piv5) leads to proteasome - mediated degradation of stat2 and stat1, respectively . However, denv ns5-mediated stat2 degradation requires an extra step as compared with paramyxovirus - induced degradation . Unlike hpiv2 v, ns5 does not mediate stat2 degradation when it is simply expressed exogenously from a plasmid . Instead ns5 has to be expressed as part of a larger precursor protein that is then proteolytically cleaved to yield a stat2-degradation - competent ns5 . During a denv infection, ns2b/3 protease cleaves ns5 away from ns4b, and it is this processed ns5 that facilitates stat2 degradation . Stat2 degradation also proceeds when an ns5 construct containing a tobacco etch virus (tev) protease cleavage site at its n - terminus is expressed in cells expressing tev protease, suggesting that it is cleavage alone and not the identity of the protease catalyzing the cleavage that determines if ns5 is able to efficiently mediate stat2 degradation . In fact, our group was able to take advantage of this unique feature to engineer an ns5 construct that would effectively mediate stat2 degradation when expressed in cells . When denv ns5 is engineered with an ubiquitin moiety fused to its n - terminus (ubiquitin - ns5), cellular hydrolases cut ubiquitin away from ns5 similarly to how the viral protease cuts ns4b away from ns5 during denv infection . When we purified ns5 from human cells expressing tagged ubiquitin - ns5, we identified a 600 kd host protein known as ubr4 . Ubr4 binds preferentially to proteolytically - processed denv ns5 over unprocessed denv ns5 but does not bind to other flavivirus ns5 proteins . Denv - mediated stat2 degradation and denv replication decrease when ubr4 levels are reduced by rna interference in interferon - competent primary dendritic cells and cell lines . However, decreasing ubr4 levels in cells that cannot produce ifn/ does not affect denv replication unless exogenous ifn/ is added to these cells . Thus ubr4 is required by ns5 to antagonize ifn/ signaling but does not appear to be necessary for other aspects of denv replication . Though ubr4 does not contain a known e3 ligase motif like a hect or ring domain, it is a member of the n - recognin / ubr family, which contains several confirmed e3 ligases . Whether ubr4 functions as an e3 ligase or as the recognition subunit of a larger e3 ligase complex is currently under investigation, but its identification as an important player in denv - mediated stat2 degradation lays the foundation for designing therapeutics that target the ns5/ubr4 interaction . The interaction between ns5 and ubr4 may also inform denv vaccine design . Live vaccines such as the truncated ns1 influenza virus mutants that are defective at evading the ifn response, have been shown to safely induce immunity in animal models . We have found that ns5/ubr4 interaction and denv - mediated stat2 degradation require amino acids threonine 2 and glycine 3 of ns5 . Mutation of these residues or others that prevent interaction of ns5 and ubr4 but leave other functions of ns5 intact could result in viruses that would be attenuated in humans due to their increased sensitivity to the effects of ifn/. Such viruses may function as effective denv vaccines . Denv is unable to replicate in wild - type mice but it can replicate in mice that have defects in ifn signaling due to the absence of ifn/ receptors or one or more stat proteins . Our group has shown that a major reason that denv replicates efficiently in human but not mouse cells is denv s ability to subvert human but not murine ifn/ signaling . Denv ns5 can bind human stat2 but is unable to bind murine stat2, and as a consequence, mouse stat2 is not degraded in denv - infected mouse cells . In a side - by - side comparison of the effects of human stat2 and mouse stat2 in stat2-deficient human or murine cells, ifn/ was able to inhibit denv replication in cells expressing murine stat2 but not in cells expressing human stat2 . This suggests that replacing murine stat2 with human stat2 in mice could potentially result in an immune - competent animal that would permit denv replication, especially as murine and human stat2 are interchangeable in the type i ifn signaling cascade . In the mouse model of piv5, transgenic expression of human stat2 permits parainfluenza virus 5 v protein to evade ifn signaling by binding stat1 . This strategy would be unsuitable for denv however, because murine stat2 would still be available to transmit signals within the cascade . Serious dengue illness occurs only in mice that lack components of both type i and type ii ifn signaling, indicating that a good mouse denv model may require modification of type ii ifn signaling components or downstream effectors in addition to replacing murine stat2 with human stat2 . Two recent papers suggest that denv tropism is also determined at the level of ifn production . Ns2b/3 protease cleaves human but not murine sting and this results in increased type i ifn production and lower denv replication in mouse vs. human cells . Thus other pathways may also need to be modified to create the ideal denv mouse model . A clonal immunocompetent mouse model would be an improvement on the currently available denv models: ag129 mice and mice humanized with cd34 human cells . Ag129 mice lack type i and type ii ifn receptors and are therefore not amenable to studying how the immune system participates in denv disease . Though humanized mice develop a human - like system, engraftment of cd34 human cells is variable and can lead to considerable variation from mouse to mouse . It is likely that a human stat2 knock - in mouse would have a functional immune system but would also permit enough denv replication to allow for the isolation of denv variants that are better at evading later blocks to viral replication . This would expedite the identification of additional determinants of denv pathogenesis thus providing new targets for rational drug design . A large portion of the denv genome is devoted to encoding proteins such as ns2b/3 protease, which inhibit ifn/ expression, and ns5, ns4b, ns2a, and ns4a, which inhibit ifn/ signaling . The ns5 protein of all flaviviruses tested so far have been shown to antagonize ifn signaling though by completely different mechanisms suggesting that the ns5 ifn signaling antagonism function arose independently several times throughout evolution . It is likely that the ns5 protein acquired the ifn signaling inhibition function in each case because the polyprotein - based strategy of flaviviral protein expression results in excess expression of ns5 even though only small amounts are needed for polymerase and transferase functions . The study of denv - mediated ifn antagonism and ns5-mediated stat2 degradation in particular has offered insights that could guide the development of an immunocompetent mouse model of dengue disease . These findings may also be useful for designing anti - denv vaccines and drugs . Continued research into the immune evasion strategies of denv is expected to yield impactful tactics for fighting denv in the coming years . (a) ifnar1/2 engagement by ifn/ leads to the activation of janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2), two tyrosine kinases that are associated with ifnar1/2 . Tyk2 and jak1 phosphorylate signal transducer and activator of transcription 1 (stat1) and signal transducer and activator of transcription 2 (stat2), which interact with interferon regulatory factor 9 (irf9) to form ifn - stimulated gene factor 3 (isgf3), which recognizes ifn - stimulated response elements (isres). Binding of isgf3 to isres leads to the transcription of ifn - stimulated genes (isgs). Several isgs, such as isg20, viperin and ifitm13, encode proteins with anti - denv activity . Ns5 inhibits ifn/ signaling by targeting stat2 for degradation while the ns2a, ns4a and ns4b proteins inhibit stat1 phosphorylation . (b) denv - mediated stat2 degradation requires proteolytic cleavage of ns5, which promotes its interaction with ubr4, a 600 kd host protein . The interaction of ns5, stat2 and ubr4 are required for ns5-mediated, proteasome - dependent stat2 degradation.
Conduct disorder (cd) and oppositional defiant disorder (odd) in children each predict adverse long - term outcomes in both mental and physical health domains (burke et al ., 2010; fergusson et al ., 2005; odgers et al ., 2008). Therefore, it is of great importance to determine if variations in brain structure exist early in the lives of these individuals . Consistent findings of morphological abnormalities would contribute to the eventual understanding of the origins and mechanisms underlying these disorders . Structural imaging studies of cd and odd during childhood would be particularly valuable for three primary reasons . First, if abnormalities are identified early in life that would suggest that at least some of the etiological influences operate early as well (e.g., genetic influences, prenatal influences, and/or early experiences). Second, because rapid neural development occurs from childhood through adolescence (casey, 2013; crone and dahl, 2012), it is important to study variations in brain structure related to odd and cd during both childhood and adolescence . Third, studies of variations in brain structure related to the disruptive behavior disorders conducted before adolescence are less subject to the criticism that the child's disruptive behaviors may increase the likelihood of traumatic brain injury and substance use, whose effects on brain structure may confound attempts to identify underlying neural mechanisms (moffitt and silva, 1988). The results of previous studies of structural variations in the brain that may be associated with odd and cd in children and adolescents have not been consistent (sterzer and stadler, 2009), but support some tentative hypotheses . In prior studies of children and adolescents, the most consistently reported structural variations associated with the disruptive behavior disorders were found in the amygdala, anterior insula, frontal cortex, and temporal lobes . Three studies reported reduced amygdala volume in adolescents with cd symptoms relative to typically developing adolescents (fairchild et al ., 2011a; huebner et al ., 2008; sterzer et al ., 2007b) a second focus of research on the disruptive behavior disorders has been on variations in the structure and function of the anterior insula, which is involved in the processing and part of a network related to empathic concern for others (decety et al ., 2008; mutschler et al ., this is relevant because deficits in empathic concern for others are common in youth with cd (frick and white, 2008; lahey et al ., 1999). Four studies have reported smaller insula volume in adolescents with disruptive behavior disorders than in healthy controls (fairchild et al ., 2011b; huebner et al ., 2008; sterzer et al ., 2007a), but one study of preadolescent boys with both conduct problems and psychopathic - like traits did not find differences in the anterior insula compared to healthy controls (de brito et al ., 2009). The medial ofc plays a role in emotion regulation and reward and punishment processing (o'doherty et al ., 2001). Several studies have linked deficits in these processes to disruptive behavior disorders in adolescents (fairchild et al ., 2013a; huebner et al ., 2008) and antisocial personality disorder (raine et al ., 2011) and psychopathy in adults (de oliveira - souza et al ., 2008; in addition, studies of children and adolescents with disruptive behavior disorders found evidence of reduced gray matter volume (gmv) in temporal regions (huebner et al . 2004), although a third study found larger volumes in a community sample of boys with cd problems (de brito et al ., 2009). Similar findings of smaller temporal lobe volumes have been reported in both incarcerated adults with personality disorders (dolan et al ., 2002) and incarcerated adult psychopaths (barkataki et al ., 2006). One functional magnetic resonance imaging (fmri) study observed less activation in the temporal cortex in violent adult offenders compared to non - aggressive offenders (raine et al ., 2001) and another documented activation deficits in antisocial and psychopathic individuals in the right posterior superior temporal gyrus during a semantic processing task (kiehl et al ., 2004). Because odd and cd are considerably less prevalent in females during childhood (lahey et al . 2004), it is important to test for differences in the neural correlates of the disruptive behavior disorders in females and males . To date, however, only one structural mri study of adolescents and young adults with cd has included both males and females (fairchild et al . Therefore, we test the general hypothesis based on previous studies that individual differences in regional gmv will be found to be associated with the disruptive behavior disorders in a sample of 911 year old girls and boys . It is important to note that our study tests these associations at younger ages than in most previous studies, which means that we are not conducting strict attempts to replicate . A stratified sample of boys and girls and their caretakers was selected based on the child's sex, race - ethnicity, and risk for high numbers of cd symptoms . A cost - efficient extreme group sampling strategy was used (preacher et al ., 2005). Based on a screening interview, children were recruited into either a high - risk stratum of children likely to meet dsm - iv diagnostic criteria for odd and/or cd and low - risk stratum of children who are unlikely to meet criteria for these diagnoses . Recruitment of children into the high - risk stratum was done with flyers calling for children with behavior problems posted in child mental health clinics and private practices . Children in the low - risk stratum were recruited from pediatric well - visit waiting rooms using a flyer calling for well - behaved children . Parents and children who consented to be screened were sequentially administered the disc predictive scale (dps) for cd, which predicts the full diagnosis of cd with high sensitivity and specificity (lucas et al ., 2001). The dps consists of 8 stem questions from the reliable and valid diagnostic interview schedule for children (disc - iv) cd module (shaffer et al ., 2000). Children were selected for the high - cd stratum if the parent alone endorsed 2 or more dps items, the child alone endorsed 3 or more items, or the parent and child collectively endorsed 3 or more items . Because odd and cd are very highly correlated during childhood (lahey et al ., 2008), participants were screened only on cd . Children were selected for the low - risk stratum if neither informant endorsed any dps cd items . To spread the distribution, children with intermediate scores of 1 on the dps were not included in the study . Selection continued until equal numbers of high- and low - risk children of each sex and race - ethnicity agreed to participate . Exclusion criteria included the presence of a pervasive developmental disorder, history of head trauma with loss of consciousness exceeding 15 min, and safety contraindications for neuroimaging . Participants who assented and whose parents gave written maternal consent were enrolled in the study, which was approved by the university of chicago institutional review board . On the day of scanning, the full disc - iv (shaffer et al ., 2000) was administered in separate rooms to the primary caregiver and to the child by trained interviewers querying symptoms during the last 12 months . A total of 169 children were screened for participation and 126 attended the mri session and gave written informed parental consent and child assent . Three of these children aborted the scan prior to completion and were excluded . After scanning, an additional 2 children were excluded due to abnormal structural scans and 12 children were excluded due to excessive movement or failure in the segmentation step of the processing . Demographic and behavioral characteristics of the 111 children included in the present analyses are shown in table 1 . Among the scanned children, 43 met criteria for a disruptive behavior disorder (i.e., they met dsm - iv criteria for odd and/or cd) in the full disc - iv interviews . Of that number, 21 met criteria for cd . The remaining 68 scanned children did not meet diagnostic criteria for either odd or cd . There were no significant differences between the three groups on sex, race - ethnicity, or maternal education . As shown in table 1, consistent with the high levels of correlations among dimensions of psychopathology in children in the population (lahey et al ., 2008), there were differences in the numbers of all dimensions of symptoms . Structural mri was acquired using a philips achieva 3 t mri scanner with a quasar dual gradient system and a 16-channel head coil at the university of chicago medical center . Images were acquired using a gradient echo, 3d t1-weighted pulse sequence (voxel size = 1 1 1 mm, tr / te = 8.1/3.7 ms, matrix size = 224 224 169, inversion time = 940 ms, and flip angle = 8). Images were first inspected by a neurologist (tz) for structural abnormalities, as well as for image artifacts and excessive motion or failure of tissue segmentation . Voxel - based morphometry (vbm) analysis was performed on the final sample of 111 children using spm8 (wellcome trust department of imaging neuroscience, london). Vbm is a voxel - wise method for comparing regional gm concentrations (ashburner and friston, 2000, 2001). Preprocessing of the t1-weighted images was done using the vbm8 anatomy toolbox (http://dbm.neuro.uni-jena.de). All t1-weighted images were corrected for intensity inhomogeneity, then spatially normalized into mni space and segmented into gm, wm, and cerebrospinal uid (csf) probability maps using the same generative model (ashburner and friston, 2005). The spatially normalized gm probability maps were then modulated by the amount of non - linear deformation, resulting in regional gmv maps . Quality assurance review of the gm tissue probability maps was done using the vbm8 tools . Total brain volume was estimated with the easy volume toolbox (pernet et al ., 2009). Following preprocessing, statistical analyses were performed in spm8 using general linear models . In the primary analyses, whole - brain analyses were conducted to test for differences in peak - level regional gmvs between (a) children who did and did not meet criteria for a disruptive behavior disorder diagnosis (odd and/or cd), and (b) children who met criteria for cd compared to children who met criteria for neither odd nor cd . Owing to the considerable range of cd symptoms within the group of children with a disruptive behavior disorder, follow - up analyses tested linear and quadratic terms for the number of cd symptoms in the scanned sample . In all models, total brain volume, age, child's sex, two dummy variables for race - ethnicity (african american and hispanic versus non - hispanic white), and maternal education were included as covariates of no interest . Maternal education was included because it is robustly associated with the child's tested intelligence (bornstein et al ., 2013; edwards and roff, 2010; ghassabian et al ., 2014). In follow - up analyses, the number of adhd symptoms also was included as a covariate of no interest to control this common correlate of cd . The critical threshold for whole - brain analyses was set at p <0.05, using family - wise error (fwe) correction for multiple testing (nichols and hayasaka, 2003). Demographic and behavioral characteristics of the sample described are in table 1 . In whole - brain analyses, there were no significant regional differences in gmv between the children with and without a disruptive behavior disorder diagnosis . Similarly, there were no significant regional differences in gmv between the 21 children who met criteria for cd and the 68 children who met criteria for neither odd nor cd . The group - by - sex interaction was not significant in any region at p <.05, with fwe correction in either comparison . Whole - brain tests of the linear term for the association of regional gmvs with the number of cd symptoms were not significant at p <.05, with fwe correction . As illustrated in fig . 1, however, the quadratic term for the association of the number of cd symptoms with gmv along the left superior temporal sulcus (sts; 63, 14, 9) was significant, f(2, 104) = 13.23, p = 0.05, k = 5, with fwe correction in whole - brain analyses, with a trend in the right sts (55, 45, 9), f(2, 104) = 10.91, p <0.001 uncorrected, k = 1515 . Furthermore, trends toward interactions of the child's sex with cd symptoms were observed bilaterally: left hemisphere, f(1, 104) = 12.65, p <0.001, k = 930, uncorrected; right hemisphere, f(1, 104) = 11.85, p <0.001, uncorrected, k = 2197 . Because such interactions with sex are theoretically important, post hoc tests were conducted in an exploratory spirit . These revealed robust significant inverse quadratic associations for cd symptoms with regions in and around the sts (left hemisphere: t = 4.95, p <0.001, fwe - corrected; right hemisphere: t = 4.67, p <0.001, fwe - corrected) in females, but no significant associations or trends at even p <.001 uncorrected in males . We repeated the analyses of associations between gmv and cd symptoms including the number of 12-month adhd symptoms as a covariate of no interest . The results were qualitatively the same as without this covariate: the quadratic term for the association of the number of cd symptoms with gmv along the left sts (63, 12, 9) remained significant, f(2, 103) = 13.61, p = 0.05, k = 5, with fwe whole - brain correction, as did the trend in the right sts (56, 45, 9), f(2,103) = 10.74, p <.001 uncorrected, k = 1453 . Unlike previous studies, we did not find significant regional differences in gmv between children with and without a disruptive behavior disorder diagnosis and in the comparison of groups who met criteria for cd versus children who met criteria for neither odd nor cd . Although there are many reasons why differences between groups that are present in the population might be detected in one study and not in another, the failure to confirm previous findings at least indicates that there is still much to learn about variations in regional gmv and disruptive behavior disorder in children . It is interesting that we found a nonlinear association in whole - brain analyses between the number of cd symptoms and gmv in and around the left sts (p <.05, fwe - corrected), with a trend toward an inverse quadratic association in the right sts . As illustrated in fig . 1, the observed quadratic association with the left sts indicates that the decline in gmv is progressively steeper at higher numbers of cd symptoms . That is, it may be that smaller gmv in the left sts region is primarily found among children with particularly high numbers of cd symptoms . It is quite plausible that variations in sts gray matter could be associated with cd symptoms in children . The sts is a component of the extended network that processes emotional information (pessoa, 2008) and is part of the human ventral attention system that detects salient and biologically important stimuli in the environment (corbetta et al ., 2008). Of potential importance to cd, the sts is involved in the perception of faces and voices (ethofer et al ., 2013) and the interpretation of the goals and intentions of others (vander wyk et al ., furthermore, functional imaging studies of mentalizing and the attribution of intentionality have revealed involvement of the posterior temporal parietal junction (tpj)/sts region (decety and lamm, 2007; pelphrey and carter, 2008). Because the sts receives information from both dorsal and ventral visual streams, it serves as an attentional hub as well as playing a pivotal role in interpreting the mental states of others (blakemore and decety, 2001; decety and cacioppo, 2012; decety et al ., this combination of recognizing the actions and intentions of other people and categorizing them as threatening and making appropriate responses are relevant to antisocial behavior . Variation in sts activation during facial emotion processing has been associated with antisocial behavior (marsh et al ., 2008; passamonti et al ., 2010). Furthermore, the sts / tpj has been implicated in generating p3 electrophysiological responses that promote attention and information processing and a large literature suggests that antisocial adolescents and adults exhibit deficits in p3 responding (gao and raine, 2009; gao et al ., 2013). In previous studies, the direction of the association of temporal lobe gray matter with antisocial behavior in children has been reported to be both positive (de brito et al ., 2009) and negative (huebner et al ., 2008; kruesi et al ., there are more consistent findings of reduced temporal lobe gray matter in antisocial male adults, but it is not clear that if reduced volume is (barkataki et al ., 2006) or is not associated with psychopathy (dolan et al ., 2002). The present findings are interesting in raising the possibility of sex differences in the association of sts gmv and cd symptoms . It should be emphasized that the sex - by - cd symptom interaction was not significant after fwe correction, but we tentatively discuss this issue because the post hoc tests revealed robust inverse nonlinear associations between the number of cd symptoms and sts gray matter bilaterally in girls (p <0.001, fwe - corrected) that were not evident in boys . If this sex - by - cd symptom interaction is replicated in future studies, it may help understand the large sex differences in the prevalence and possibly the etiology of cd behavior (moffitt et al . Gender paradox in many disorders with such unequal sex ratios: the gender with the lower prevalence also tends to be more seriously affected . The present findings raise the important possibility that more pronounced structural abnormalities in the sts may be needed to overcome other factors that protect girls from developing cd symptoms during childhood . If boys are less protected from social influences to engage in childhood antisocial behavior, they may be more likely than girls to develop cd symptoms in the absence of reduced gmv in the sts . A related possibility is that a sex difference in the development of temporal lobe structures from childhood through adolescence and into adulthood (hu et al ., 2013; mcclure et al ., 2004) could result in temporal lobe structures being related to antisocial behavior in different ways at different ages in female and male children . For example, it is possible that the reductions of gmvs that have been documented in previous studies of antisocial male adolescents and adults only emerge after childhood . If the sex - by - cd symptom interaction is substantiated in future studies, longitudinal imaging studies beginning in childhood will be needed to determine if this sexual dimorphism is observed across age within the same sample and accounts for some of the sex differences in adolescent antisocial behavior . Such findings would not necessarily rule out other explanations, of course, such as sex differences in socialization (keenan and shaw, 1997). Although maternal education is robustly predictive of her child's intelligence (bornstein et al ., 2013; edwards and roff, 2010; 2014), our findings may have differed had child intelligence been directly measured in the present study (wallace et al ., 2014). In addition, our focus on gray matter volume is an important limitation of the present study . Gray matter volume is a function of both cortical surface area and thickness and there is emerging evidence that cortical surface area and thickness have different influences and correlates (winkler et al ., 2010). For example, one study failed to find differences in gmv between children with a disruptive behavior disorder diagnosis and healthy controls, but found reduced cortical thickness in the disruptive behavior disorder group (fahim et al ., similarly, two studies found reduced cortical thickness, but not surface area, in the superior temporal cortex in youth with cd (hyatt et al ., 2012; wallace et al ., 2014). Thus, the present study may have been more revealing had these components of gmv been distinguished . The present findings were consistent with previous findings of an association between high levels of cd symptoms and reduced gray matter volumes in temporal regions, although this association may have been stronger in girls . The present findings did not support previous findings of reduced gray matter volumes in the anterior insula, amygdala, and frontal cortex in mostly older children and adolescents who met diagnostic criteria for odd and/or cd relative to healthy comparison children.
Reversal or shrinkage of early ischemic injury demonstrated by diffusion - weighted mri (dwi) is clinically uncommon, even after early thrombolytic reperfusion therapy [1, 2, 3, 4, 5]. The dwi - defined early ischemic injury physiologically represents an irreversible ischemic core and a potentially reversible surrounding area . Recently, we experienced the case of a nearly complete reversal of a hyper - acute large ischemic injury on dwi . A 67-year - old previously healthy woman suddenly developed stupor and left hemiparesis, and arrived at our hospital 45 min after symptom onset . Upon presentation, the national institute of heath stroke scale (nihss) score was 12 points, and baseline brain ct was perfectly normal . Dwi (single - shot echo - planar, b = 1,000 s / mm, tr = 8,000 ms, te = 80.8 ms, slice thickness = 6 mm) performed on the 1.5 t mr scanner (signa 1.5 t; general electric, usa) imaged 78 min after onset demonstrated a large high - signal intensity area in the right hemisphere with a dwi - aspects (albert stroke program early computed tomography score) of 5 points, and mr angiography (mra) showed an occlusion of the right middle cerebral artery trunk (fig . Perfusion mri was not performed . On the apparent diffusion coefficient (adc) map, the mean ratio of the adc value of ischemic to unaffected hemispheres was 0.86 (range 0.780.92) within the five dwi - aspects - negative ischemic areas, and the mean adc ratio was 0.99 (0.981.00) within the remaining five dwi - aspects - positive non - ischemic areas . Accordingly, the patient received intravenous thrombolysis with tissue plasminogen activator at a lower dose of 0.6 mg / kg alteplase on the japanese guideline . The state of health of the patient was improved, and the nihss score 24 h later was of 0 points with minor left - sided numbness . The second dwi (b = 1,000 s / mm, tr = 5,000 ms, te = 55 ms, slice thickness = 5 mm) performed on the 3 t mr scanner (achieva 3 t; phillips, the netherlands) imaged 30 h later showed a nearly complete reversal of the large high - signal intensity area demonstrated by initial dwi, and there were very small high - signal intensity spots at the cortical surface (fig . The mean adc ratio was 1.00 (0.991.02) within the five areas of the initial dwi - aspects - negative ischemic territory and 0.97 (0.881.01) within the remaining five initial dwi - aspects - positive non - ischemic areas . The systemic work - up including cardiac and hematological profiles revealed the presence of patent foramen ovale and venous thrombus in her leg, suggesting a paradoxical brain embolism as an etiological mechanism for this stroke . The patient received anticoagulation with warfarin for secondary prevention . At 3 months, clinical outcome was a modified rankin scale score of 0, and only minor final infarction was localized at the cortical surface on the fluid - attenuated inversion recovery (flair) image (tr = 11,000 ms, te = 125 ms, slice thickness = 5 mm) (fig . In this patient, after early thrombolytic reperfusion therapy, the initial dwi - defined large hemispheric ischemic injury showed nearly complete reversal on the follow - up dwi, and very small thrombus - fragmented ischemic areas after complete vessel recanalization were localized at the cortical surface of the terminal zone of the initially occluded middle cerebral artery . A limitation of this case study was the subtle low imaging quality of the hyper - acute dwi, due probably to body motion in acute confusion and to the simple acute stroke mri protocol to minimize scan time in the emergency setting . To assess the ischemic injury more accurately, an advanced 3-tesla mr scanner was utilized at the later scans, although a difference of mri scanner between hyper - acute and the later phases may affect the precise comparison of ischemic injury . Since a recent study reported that standard 1.5-tesla mr scanner, rather than 3-tesla mr scanner, provided a lower false - positive and false - negative rate for detecting dwi - defined early ischemic injury within 6 h after stroke, hyper - acute dwi imaged on a 1.5-tesla mr scanner might have been an optimal selection as acute stroke mri protocol in this case study . The latest meta - analysis reported that the rate of complete or partial reversal of ischemic dwi lesions was surprisingly high by an average of 24%, varying from 0 to 83% probably due to the variable definition of the dwi reversibility . Time of acute dwi scan from stroke onset, as well as time and type of sub - acute to chronic scans such as dwi, t2- or flair image to assess the final infarct area, may affect frequency and size of the dwi reversibility . True reversal of a dwi abnormality, defined by eliminating the false - positive reversal effect, was uncommon at only 7% in patients receiving early intravenous thrombolysis . In this patient, nearly complete reversal of the initial dwi - defined large ischemic injury persisted on the 3-month follow - up flair image . A previous study quantitatively measured severity or depth of ischemic injury by adc values to define the threshold of the infarct process . In the dwi - defined ischemic area, an adc value below 80% of the unaffected area and no reperfusion were the factors of dwi irreversibility, but the exact threshold of the adc value could not be determined . In this patient, an adc decline with an average of 86% in the dwi - defined ischemic area and early successful reperfusion may largely account for complete and sustained reversal, associated with a favorable clinical outcome . In conclusion, hyper - acute large ischemic injury demonstrated by dwi may very rarely represent a nearly perfect reversible area, and it may be a therapeutic target.
According to the statistical analysis of the international society of heart and lung transplantation, survival after lung transplantation systematically improves . 88% of recipients survive three months after operation, 79% survive for one year, and about 50 - 60% of recipients survive five years after transplantation . The most common indication for lung transplantation is chronic obstructive pulmonary disease (copd) (34%). The major early causes of death are graft failure and non - cmv infections, bronchitis obliterans syndrome (bos), chronic lung rejection, and graft failure after one year . A 58-year - old female with hyperthyroidism, osteoporosis, and copd in the fourth stage was classified for lung transplantation in 2011 . Additional tests demonstrated pao2 (partial pressure of oxygen) 61.2 mmhg, paco2 (partial pressure of carbon dioxide) 44 mmhg (on oxygen therapy 2 l / min), fev1 (forced expiratory volume in 1 second) 0.44 l 19.7%sd, fvc (forced vital capacity) 1,38,8% sd, 6-mwt (six - minute walk test) 174 metres, rvsp (right ventricular systolic pressure) 36 mmhg, and pra (panel reactive antibody) 3% . After four months of expectation a 57-year - old female donor, who died because of cerebral haemorrhage and was ventilated for 48 hours with pao2/fio2 360 mmhg, was identified . Both women were characterised by comparable growth and identical blood group . The day after donor identification the transplantation was performed . For this purpose the right pulmonary vein cuff proved to be very short and tight, so it was decided to implant the left donor lung in the recipient's right emphysemal lung locus . Elements of the recipient's right lung hilum were dissected, cut distally with clamping of the pulmonary artery and pulmonary veins cuff . After preparation the left donor's lung was implanted in the contralateral position . In conjunction with different bronchus diameters the donor bronchus was telescopically placed in the recipient's bronchus and stitched with single absorbable sutures 3 - 0 . Two drains were inserted in the pleural cavity, and closure of the thorax was carried out . Metal suturing of the sternum and the suturing of the periosteum of the fifth rib and layered lid suture were performed (fig . The patient was transferred to the intensive care unit (icu), where she stayed for eight days . Because of retrospectively positive cross - match it was necessary to implement five automatic plasmapheresis and to apply human immunoglobulin and rituximab to diminish concentration of lymphocytotoxic antibodies against hla antigens . On the third day after transplantation the primary graft dysfunction and rising emphysema of the recipient's own lung shifted the mediastinum towards the graft side . As well as standard treatment, c1-esterase was provided to deplete the extracapillary exudation, and the operative (lung volume reduction surgery lvrs) reduction of emphysemal changed lung tissue was pursued with complication of prolonged air leakage (fig . The patient needed infusion of 16 units of fresh frozen plasma (ffp), 2 units of packed red blood cells (prbcs), and 100 ml of 20% albumin . The right lung drainage was removed after seven days and the left one after 35 days . On the 44 day after transplantation the patient was discharged from hospital with the following study results: po2 68.9 mmhg, pco2 44.0 mmhg (without oxygen therapy), fev1 1.12 - 50.5%, fvc 1.38 - 52.2%, 6-mwt 246 metres, and with recommendation of taking immunosuppressing medications such as: 50 mg of cyclosporine twice a day, 1000 mg of mycophenolate mofetil twice a day, and 10 mg of prednisone once a day . 3). It was histopathologically confirmed and antibodies against donor antigens were detected (anti - hla class ii 158.57 - 701.89 mfi). On the third day the therapy with methylprednisolone was pursued . In chronically treatment instead of cyclosporine the tacrolimus of 0.5 mg a day was implemented . Additionally, the hospitalisation was complicated by moraxella catarrhalis infection, so amoxicillin with clavulanic acid was applied . Diagnostic tests performed meanwhile showed transient renal failure, ion disorders, hypoproteinaemia, and leukopaenia . At the turn of fifth and sixth month after transplantation the next complications occurred: bronchitis caused by corynebacterium pseudodiphtericum, and escherichia coli urinary tract infection, both treated with amoxicillin and clavulanic acid, and a second episode of acute graft rejection . During the next two months e. coli sepsis was seen and was treated with ceftazidime . Then the cytomegalovirus disease treated with ganciclovir, pulmonary embolism, and anxiety disorder treated with opipramol and venlafaxine occurred . On account of leukopenia 2900/l after the next month the third episode of acute graft rejection appeared, following bacterial infection caused by pseudomonas aeruginosa cured with ciprofloxacin and ceftazidime and bilateral otitis with sinus and temporal bone polyposis development, hence clindamycin and budesonide inhalation were applied . Because of hypogammaglobulinaemia it was decided to assign human immunoglobulins every three weeks . During the 11 and 12 month after the transplantation (fig . 4) the next bacterial infections caused by acinetobacter baumanii, proteus mirabilis, candida albicans, c. glabrata and aspargillus species ensued . The therapy contained cefuroxime, imipenem, ampicillin with sulbactam, amikacin, amphotericin b, itraconazole, and colistin . Meanwhile drug - induced diabetes with rapid decompensation occurred, which initially needed glimepiride and then insulin therapy . Later, renal failure and a fourth episode of acute lung rejection appeared . Between the 14 and 16 month after lung transplantation the fifth episode of rejection appeared, renal failure deepened, and destabilisation of diabetes occurred . The cytomegalovirus disease activated and valganciclovir was used in therapy, but the leukopaenia deteriorated and it was necessary to reduce the dose of the drug . In addition, acinetobacter baumani, klebsiella pneumoniae, and candida albicans infections appeared . In therapy the ceftazidime, ciprofloxacin, ampicillin with sulbactam, colistin, metronidazole, tazobactam, itraconazole, and nystatin were used . At this time unfortunately, the psychological state of the patient gradually worsened, and she manifested symptoms of depression . Collateral complication appeared as posttraumatic haematoma of the shin, which required vac - therapy (vacuum assisted closure vac). 498 days after transplantation the patient died due to symptoms of sepsis after unsuccessful reanimation . Two drains inserted in right pleural cavity and catheter in right carotid internal vein are visible chest x - ray performed after lung volume reduction surgery (lvrs) chest x - ray performed during first episode of acute rejection chest x - ray performed one year after transplantation lung transplantation in medically incurable patients is the only method that gives a chance to lengthen their lives . Currently it is recommended that the cross - match be pursued before performing the transplantation to avoid such a situation, although there are some scientific reports discussing the primary existence of these antibodies in the recipient's organism or their appearance as the consequence of transplanted organ dysfunction . There are some case reports that adduce false positive and false negative pre - transplant virtual cross - matches . However, virtual cross - match should be considered as a routine technique used before transplantation procedure in poland . Virtual cross - match is the admitted method of prediction if the recipient produces hla - antibodies . An appropriate computer program determines every hla antigen immunogenicity that is incompatible with full recipient hla haplotype as an inconsistent immunogenic loci (miss - match amino acids eplets mmep). Safe immunogenic incompatibility that does not result in humoral response can also be predicted with this method . It is a huge advantage in patients who need multiple transplantations . Extending the scope of immunological selection to hla - cw, dq, dp increases the effectiveness of virtual cross - match (91%) in comparison to serological selection on hla - a, b, dr used in poland . Difficulties in proper donor selection are related with imperfection of chemical reagents and antibody detection techniques . In the first case the chance for transplantation is wasted, in the second the patient is exposed to serious postoperative complications . Furthermore, the role of antibodies against the host's own (personal) antigens in pathogenesis of chronic dysfunction of transplanted organs is discussed . However, no matter which hypothesis is accepted, only quickly implemented therapy can improve the patient's condition . There are some ambiguous reports about monoclonal antibodies although there are some indications that such treatment can be effective [6, 7]. We proceeded with mandatory cross - match before lung transplantation in every recipient with pra> 0% . Also, the age of qualified donors is limited to 55 years old, whereas the criterion of pao2/fio2 was raised to> 400 mmhg . The recommended operation technique is bilateral lung transplantation because of the reservoir of microorganisms in the recipient's own lung, so nowadays in such a clinical situation probably reconstruction of the donor's right pulmonary vein cuff would be performed . Additionally, lvrs, according to present knowledge, would not be pursued, because the shading of the graft after transplantation was caused by primary graft dysfunction and not by the recipient's own lung . Finally, meticulous training of the transplantation team in surgical techniques should be continued, which is achieved in our case by practicing microsurgical techniques on rats and by using a cadaver workshops.
Traumatic brain injury (tbi) affects about 1.7 million americans annually (cdc, 2010), with ~80% of the cases categorized as mild (mtbi) (faul et al ., 2010). Computerized tomography (ct) and conventional magnetic resonance imaging (mri) often appear normal without anatomical lesions in mtbi (hughes et al ., 2004; bazarian et al ., 2007) and have been considered less sensitive than more advanced forms of mri to diagnose mtbi or monitor the course of injury . Therefore, mtbi is generally diagnosed on clinical assessment, based upon reported history of loss of or impaired consciousness, post - traumatic amnesia, or post - concussion symptoms (pcs) (e.g., headaches, dizziness, fatigue, irritability), and cognitive / memory complaints . The underlying pathologic mechanisms in mtbi are still unclear although it has been postulated that stretch - induced axonal injuries may be the cause (blumberg et al ., 1995). Advances in mri - based neuroimaging techniques, such as diffusion tensor imaging (dti), magnetization transfer ratio (mtr), magnetic resonance spectroscopic imaging (mrsi), and perfusion imaging, have brought new potential to mtbi diagnosis (shenton et al ., 2012). Dti investigation of mtbi patients has attracted researchers because this imaging modality is particularly sensitive to microscopic white matter (wm) changes and may be able to detect diffuse axonal injury in mtbi (shenton et al ., 2012; bigler, 2013; hulkower et al ., 2013; fox et al ., 2013; ling et al ., 2013; kou et al ., 2013; hasan et al ., dti is based on diffusion of tissue water molecules and by exploiting the anisotropic nature of diffusion it is possible to gain information about the microstructural organization and integrity of wm fiber tracts that interconnect various brain regions (mori and zhang, 2006). The most commonly used dti - derived measures are the fractional anisotropy (fa) that reflects diffusion anisotropy and mean diffusivity (md) that represents average diffusivity and axial diffusivity (ad), and radial diffusivity (rd). Together, these dti measures could serve as markers of tissue integrity (pierpaoli and basser, 1996). Previous dti reports on mtbi have produced somewhat inconsistent results (see for example, the review by shenton et al ., 2012). Reduced fa and elevated md in various wm regions in the acute and chronic phase of mtbi were reported (see for example, toth et al ., 2013). Increased fa and md were also reported both in acute and subacute phases (bazarian et al ., 2007; wilde et al . Both increased and decreased fa were reported in the same patients (kou et al ., 2013). A recent study reported changes only in md in certain wm tracts (hasan et al ., 2014). Finally, no changes in fa were observed in concussed individuals at about 30 days post - injury (zhang et al ., 2010). Since myelin represents major macromolecular pool in brain, it is generally thought that mtr is sensitive to the state of myelin (see for example, mcgowan, 1999) and is therefore an indirect measure of myelin integrity (pike et al ., 2000; mottershead, 2003). Reduction of mtr was reported in some wm regions in mtbi and in some cases significant correlations were found between regional mtr and neuropsychological performance (mcgowan et al ., 2000). Histogram based analysis showed reduced whole brain mtr in moderate tbi and mtbi subjects with persistent pcs (hofman et al ., 2002). Mri is an excellent modality for estimating global and regional atrophies in mtbi and for following their longitudinal evolution . There are only a few studies that investigated atrophy in mtbi (ling et al ., 2013; cohen et al ., 2007; lannsj et al ., 2013; ross et al ., 2012; zhou et al ., 2013). These studies suggested measurable atrophy in mtbi around 1 year after the insult (zhou et al ., 2013). Regional atrophy was also observed in the anterior cingulate, cingulate gyrus, and the right precuneal gray matter (gm) (zhou et al ., 2013). These observations are similar to those reported earlier (mackenzie, 2002). In a 2 year follow - up study, progressive atrophy was also observed in mtbi (ross et al ., 2012). Most of these studies, however, were based on relatively small sample size with variable post - injury scanning times . Also, the earliest time frame at which atrophy could be detected following mtbi is unknown . Metabolic disturbance is postulated in regions of contused brain (giza and hovda, 2001). Magnetic resonance spectroscopy (mrs) measures brain metabolites that reflect local neuronal integrity and cell membrane turnover (narayana, 2005). Application of mrs to mtbi was recently reviewed (lin et al ., 2012; gardner et al ., 2014). Because mtbi induces a cascade of changes in brain metabolites, including neuronal and axonal loss, mrs has the potential to improve our understanding of the underlying metabolic disturbances in mtbi (cohen et al ., 2007; lin et al ., 2012; vagnozzi et al ., 2008; henry et al ., the major peaks observed in proton mrs of brain include n - acetylaspartate (naa) + n - acetyl aspartyl glutamate (naag) (it is generally difficult to resolve these resonances and naa + naag is referred to as naa), creatine (cr), choline (cho), and myoinisotol (mi). Creatine resonance has contributions from creatine and phosphocreatine and elevated cr levels may represent gliosis . Choline resonance has contributions from multiple molecules that include phosphorylcholine, glycerophosphorylcholine, and choline plasmalogen, and a minor contribution from acetylcholine and choline . Choline peak reflects cell membrane metabolism and elevated cho concentration represents heightened cell membrane turnover as seen in demyelination, remyelination, inflammation, and gliosis . Myoinisitol is thought to be glial specific and is also a precursor of phospholipid membrane constituents and its concentration is affected by the formation and breakdown of myelin (reviewed in sajja et al ., 2009). The results for mrs in mtbi, however, are not always consistent (reviewed by gardner et al ., 2014). Based on this brief description one may conclude that each of the mri modalities is sensitive to different aspects of tissue pathology . It is possible to improve the pathologic specificity in mtbi by using multi - modal mri . While there have been many independent studies using any one or two of these modalities (reviewed in shenton et al ., 2012; lin et al ., 2012), we are unaware of the application of all these techniques to the same mtbi cohort . The main objective of this study was to perform comprehensive multi - modal mri in mtbi and orthopedic injured (oi) controls . First, in almost all the previously reported studies scans were acquired days or even weeks post - injury for determining the acute changes in mtbi . It is not clear what changes occurred in the first 24 h after the injury . This work was approved by the institutional review boards (irbs) at participating institutions and the human research protection official's (hrpo) review of research protocols for the department of defense . All procedures were compliant with the health insurance portability and accountability act (hipaa). The project reported here is part of a larger study of mtbi, supported by the department of defense, where a consecutive series of civilian patients was recruited prospectively with either mtbi or minor orthopedic / extremity injuries from the emergency departments (eds) of two level 1 trauma centers and one level 3 community hospital in a large ethnically - diverse southwestern metropolitan area . Initial and follow - up assessments of the oi subjects provided comparative data from individuals with demographics and risk factors similar to the mtbi subjects . The definition of mtbi used in this study followed the guidelines of the department of defense (assistant secretary, 2007) and the american congress of rehabilitation medicine (kay, 1993). Subjects were included irrespective of their gender, race, and ethnicity and were recruited by healthcare professionals (rn, md, emt - p) who had clinical experience with brain injury patients, knowledge of research, and excellent interpersonal and problem - solving skills . Screening occurred through review of data in the eds electronic healthcare system (ehs), consultation with ed staff, and subject interviews . Special permission was obtained from the institutional irbs to administer the galveston orientation and amnesia test (goat) prior to obtaining informed consent to identify cognitive impairment that would preclude provision of informed consent . Subjects had to score 75 on the goat (levin et al ., 1979) to provide informed consent; if a subject's score was 74 or lower, the plan was to obtain informed consent from a legally authorized representative (lar). There have been no scores below 75, so all enrolled subjects have provided written informed consent . Inclusion criteria for both groups (mtbi and oi) included age 1850 years, injury occurring within the preceding 2448 h, and no requirement for hospitalization for the injury for which the participant was enrolled . For mtbi subjects, inclusion criteria also required the presence of a head injury (documented in medical records and/or verified by witnesses), glasgow coma scale (gcs) score between 13 and 15, loss of consciousness for <30 min (including 0 min), post - traumatic amnesia <24 h (including 0 min), and a negative head ct scan . Inclusion criteria for the oi comparison group included an abbreviated injury severity (ais) score <3 for an extremity or pelvis injury, with no head injury present . Appropriate candidates with orthopedic / extremity injuries that also sustained a head injury were enrolled in the mtbi group . Exclusion criteria for both groups included ais> 3 for any body part, history of significant preexisting disease (e.g., psychotic disorder, bipolar disorder, ptsd diagnosed by a psychiatrist or psychologist, past treatment for alcohol dependence or substance abuse), blood alcohol level> 80 mg / dl at the time of consent, documentation of intoxication, left - handedness, and contraindications for mri (including claustrophobia and pregnancy). Previous head injury requiring hospitalization or ed treatment was also an exclusion criterion for all subjects . All mri scans were performed on a philips 3 t scanner using an eight channel receive - only head coil . The mri protocol included acquisition of 3d t1-weighted images (t1 images for brevity) using the magnetization prepared rapid acquisition of gradient echoes (mprage) sequence for spatial normalization and morphometric measurements, 3d fluid attenuation by inversion recovery (flair) for visualizing lesions, if present, 2d t2-weighted gradient echo for visualizing hemorrhage, 3d gradient echo sequence for mtr measurements for assessing the state of myelin, and 2d mrsi for metabolite mapping, and a single - shot echo - planar imaging (epi) for acquiring diffusion weighted imaging (dwi) for calculating the dti measures . All mri data were automatically assessed for image quality as described elsewhere (narayana et al ., 2012; hasan et al ., 2014) and scans with inadequate quality were discarded from further analysis . Consistent performance of the scanner and its temporal stability are critical for longitudinal studies (hasan, 2007). The quality assurance (qa) issues are addressed by implementing the american college of radiology (acr; http://www.acr.org/quality-safety/accreditation/mri) recommended qa program that measures signal - to - noise ratio, field uniformity, gradient linearity, image distortions and ghosting using the vendor provided phantom every morning . In addition, as described elsewhere (hasan et al ., 2014), dti data was acquired monthly on a homogenous spherical water phantom that was kept at a temperature between 18 and 20 c (temperature in the scanner room), maintained by the central air conditioner . The dti data was analyzed using the procedure described elsewhere (hasan et al ., 2014). Briefly, dti data was acquired on a spherical water phantom almost monthly over 4 years (47 time points). The mean fa and md values and their standard deviations were automatically calculated from an roi of 21 21 voxels at the isocenter of the magnet . The dwi images in the dicom format were transferred from the mri scanner to a local linux computer (xeon 2.8 ghz 4-core cpu with 24 gb memory) and were converted to neuroimaging informatics technology initiative (nifti) format using dcm2nii file converter from the mricron software (http://www.cabiatl.com/mricro/mricron/dcm2nii.html). The brain was extracted from the images using the brain extraction tool (bet) software from fsl toolbox (http://www.fmrib.ox.ac.uk/fsl/bet2/index.html). Eddy - current correction was performed in fsl by aligning all the dwi images to the b0 volume (images acquired without any diffusion weighting) using affine transformation with 12 degrees of freedom . The dti measures (fa, md, and the three eigenvalues, 1, 2, and 3) were then calculated using the fdt software from the fsl toolbox . The dti data was analyzed using tract based spatial statistics (tbss) (smith et al ., 2006). The identification of the individual tracts was verified using the mri atlas of human white matter (oshi et al ., 2010). Standard tbss analysis steps as recommended by the developer of tbss were followed . The fsl - provided fa template (fmrib58_fa; smith et al ., 2006), which was derived from healthy controls and its derived skeleton in the mni space were used . Randomize test on skeletonized dti images was conducted with threshold - free cluster enhancement (tfce) option and 5000 runs . Statistical results were generated both with and without the family - wise error (fwe) correction for multiple comparisons . Images acquired with (msat) and without (m0) the off- resonance rf pulse were stripped of extrameningeal tissues using bet from the fsl toolbox . Bias field correction was applied using the nt4 module from advanced normalization tools (ants) software package (http://sourceforge.net/projects/advants). The corrected msat and m0 images were non - linearly co - registered to the mni152 1 mm t1 template (included in fsl toolbox) using the symmetric inverse consistent diffeomorphic registration from the ants software tensor based morphometry (tbm) was used for estimating regional and global atrophy . For tbm analysis, the t1 images were non - linearly registered to the mni152 template using symmetric inverse consistent diffeomorphic registration from the ants software . The output of the affine and diffeomorphic transformation files was combined to generate the composed transformation . The jacobian determinant (jd) maps were constructed from the composed transformation of the subject - to - template registration (leow et al ., 2006). The jds were normalized to compensate for differences in the brain size and logarithmic transformation was applied (tao et al .,, statistical tests were performed on the jds using two - sample t - test model in spm8 . Paired t - tests were also performed on the subjects who had both initial and follow - up scans . For 2d csi a slab of 60 mm 60 mm 15 mm was placed in the centrum semiovale region . Great care was taken in placing the csi slab across all subjects and different time points by using various land marks such as ac pc plane and location relative the lateral ventricles . Initially all the individual mrs volumes were reconstructed in the subject s native space . For group analysis, each mrsi slab was linearly registered to the t1 scan using rigid body registration with six degrees of freedom . Then the mrs volume in the native space was warped to the mni space by applying the deformation field obtained from t1-to - mni152 non - linear registration . In the group analysis only those voxels that were common for all the subjects following transformation to the mni space, as determined by their spatial coordinates, were included . The resulting common mrs volume contained both cortical and sub - cortical regions and contained both gm and wm tissues . In order to conduct comparison on homogenous structure and tissue types, the mrs volume was sub - divided into 12 smaller volumes - of - interest (voi), 6 in each hemisphere, with each region containing only one brain structure and tissue type (sfg superior frontal gyrus, mfg superior frontal gyrus, jlc juxtapositional lobule cortex, cg cingulate gyrus, pcg paracingulate gyrus, wm white matter). Only the center 6 6 voxels (each voxel measured 10 10 15 mm) from the 2d csi slab were used since they had the highest spectral quality . Metabolite concentration ratios naa / cr, cho / cr, and mi / cr were automatically estimated with the lc model software (http://www.s-provencher.com/pages/lcmodel.shtml). The default basis set for 35 any estimated ratio with greater than 20% sd (cramer rao lower bounds) were considered to be unreliable and deleted from the data analysis . Based on the daily qa measurements, the performance of the scanner either met or exceeded the acr recommendations and vendor's specifications . The results of the dti analysis were reported earlier (hasan et al ., 2014). The average sd values of md and fa over the 4 year period were (1.88 0.16) 10 mm / s and (0.015 0.002), respectively . Linear regression of the temporal changes in md and fa did not show any trend, suggesting the absence of systematic drift with time . The mechanism of injury in both mtbi and oi subjects is summarized in table 2 . Over 90% of mtbi subjects had visible evidence of head trauma associated with multiple mechanisms, in the form of bruising / contusions / abrasions to the head, scalp, or face (84%) and scalp lacerations (44%), of which 54% (24/44) required sutures . Subjects in the oi group explicitly stated that to the best of their recollection, they did not hit their head during the event that caused their injury, they had no visible signs of injury to the head (i.e. Bruising, abrasions, contusion, tenderness, swelling, or scalp lacerations with or without sutures required), and they reported no loc or pta . Sixty - two mtbi subjects (30.4 8.8 years age, range 1950 years; number of males = 43; education = 13.6 2.4 years) and 59 orthopedic controls (age = 29.2 9 years, range 2051 years; number of males = 45; education = 13.5 2.9 years) were included in this study . All but two subjects had a gcs score in the ed of 15, with the other two having a gcs score of 14 . Among the mtbi subjects who lost consciousness after injury, the average loc duration was 3.95 5 min (range 120 min). The initial post - injury scan times for mtbi and oi were 25.5 12.26 h (range 5.846 h), and 27.1 13.70 h (range 0.356 h), respectively . The corresponding follow - up scan times were 97.9 17.57 days (range 83.3202 days) and 96.7 9.26 days (range 82.9126.9 days), respectively . It should be pointed out that the range for the follow - up for mtbi subjects may have been skewed since one subject was scanned 202 days after injury . The number of mtbi and oi controls who completed the initial scans was 56 and 54, respectively . The corresponding numbers at the follow - up were 29 (age 29.94 8.22 years, range + 18.949.7; number of males 18) and 47 (age 29.48 9.03 years; range 20.2550.75 years, number of males = 36). Mtbi subjects had the option to participate in a phase ii drug trial (21/65 or 32%) or enroll only in the testing / imaging portion of the study (44/65 or 68%). Those mtbi subjects in the drug trial were not included in the analysis of the follow - up scans, as the investigators are still blinded to which subjects were receiving active drug treatment and which subjects were receiving placebo treatment . This was done to ensure that any observations from this analysis would not be due to or influenced by the study drug . Therefore, the number of mtbi subjects that were included in the final analysis was approximately 50% of the oi subjects . There were no significant female / male ratio (p = 0.45; test) and age differences between the mtbi (56 subjects) and oi (54 subjects) control groups (p = 0.72; t - test) at the initial study . The follow - up mtbi (29 subjects) and oi (47 subjects) cohorts also did not differ either in the gender ratio (p = 0.3) or age (p = 0 . Two t1, two mtr and one dti image volumes were excluded due to poor image quality that include poor snr and motion related artifacts . Fourteen mtr image volumes were excluded due to motion between acquisition of msat and m0 images . Mtr is based on subtraction of two images and is therefore more prone to motion artifacts . Three dti image volumes were excluded due to inconsistent protocol used in the early stage of the study . The final number of mtbi and oi subjects included in the analysis for each mri modality is summarized in table 3 . 1 shows the t maps of md that shows significant differences between the mtbi and oi groups at the initial scan (fwe corrected; p <0.05) superimposed on the wmfa skeleton . As can be seen from this figure, md was significantly higher in the mtbi relative to oi subjects in several wm regions that include internal capsule (ic), external capsule (ec), superior corona radiata (scr), anterior corona radiata (acr), posterior corona radiata (pcr), inferior fronto - occipital fasciculus (ifo), inferior longitudinal fasciculus (ilf), forceps major and forceps minor of the corpus callosum (cc), superior longitudinal fasciculus (slf), and cortical spinal tract (cst). A number structures showed differences in the dti measures at p <0.05 but did not survive the fwe correction . For example, md was observed to be higher in the mtbi relative to oi in the ic, ec, scr, acr, pcr, ifo, ilf, forceps major and forceps minor of the cc, slf, and cst in both hemispheres . Increased md was also seen in the genu, body and splenium of the cc . Increased ad (1) and rd ((2 + 3) / 2) were observed in some of the regions where increased md was also found (scr / pcr, plic, ifo and acr; p <0.05). On the tbss analysis none of the mri measures on the follow - up scans, either in the mtbi or oi group, showed significant changes from baseline with fwe correction . Without fwe correction in the mtbi group md decreased at follow - up in the wm regions that included plic, ec, slf, acr, pcr, scr, ifo and cc (p <0.05). Ad also decreased in regions that included ec, slf, acr, scr and cc) and rd decreased in plic and ifo(p <0.05). As in the case of initial scan, the effects appeared to be significant and more widespread in the right hemisphere . In the oi controls, tbss analysis found no significant difference in any of the dti measures between baseline and follow - up even without the fwe correction . After registering all the subjects' mtr image volumes in the mni space, voxel based analysis was conducted to detect differences between mtbi and controls at the initial scan and also between the initial and follow - up scans in both mtbi and oi controls . The jacobian determinant (jd) maps constructed from the t1 images were used for measuring the regional volumetric changes in the brain . As in other analyses, we investigated the baseline volumetric differences between mtbi and control subjects and volumetric changes in the mtbi and oi subjects between initial and follow - up scans . With fwe correction statistically significant clusters (10 voxels) the 2dcsi slab was placed about 1 cm above the lateral ventricles, and parallel to the anterior posterior commissure line (fig . 2). The ten saturation bands for outer volume suppression are shown in blue in fig . The individual voxels (10 10 15 mm) are shown in red in this figure . The line width for the water peak from the entire spectroscopic voi was (11 2.4) hz . The line width of naa, for example, from individual voxels was (7 1.1) hz . The snr in the water suppressed spectrum in each voxel was 45 7 . The spectra from the whole slab (average spectrum) along with representative spectra from four different voxels from one patient are also shown in this figure, the minimal lipid contamination and excellent spectral resolution can also be seen on this figure . 3a) along with the parcellation of the common mrs volume into 12 regions (fig . 3b), with each region containing only one brain structure and tissue type (sfg superior frontal gyrus, mfg superior frontal gyrus, jlc juxtapositional lobule cortex, cg cingulate gyrus, pcg paracingulate gyrus, wm white matter). The results of the metabolite ratios at the baseline and follow - up for both cohorts are summarized in fig . Cho / cr ratio did not show significant differences between the mtbi and the control subjects on the initial scan in any of the voxels, nor did this ratio significantly differ between initial and follow - up scans in the mtbi group . Mi / cr ratio, on the other hand, showed lower values in the mtbi than in the comparison subjects on the initial scan in jlc, cg, and pcg, all in the right hemisphere (p <0.05). The mtbi cohort naa / cr showed lower value on the initial scan compared to follow - up in jlc in the right hemisphere (p <0.05). . We also analyzed the longitudinal changes by considering the ratio of the follow - up scans and the corresponding initial scans for each subject (data not shown). This strategy eliminates the inter - subject variation in the metabolite ratios and improves the statistical power . We did not include the analysis of the glutamate + glutamine peaks in this analysis because their crlb was greater than 20 in majority of the patients . To the best of our knowledge, this is the first multi - modal mri study on mtbi subjects . A novel feature that distinguishes this study from others is that the scans were performed at approximately 24 h after injury in ct - negative mtbi patients . In addition, the same cohort was rescanned at about 3 months post - injury . Furthermore, great care was taken to eliminate confounding factors such as alcohol and drug abuse, history of hospitalization for previous tbi, and pre - injury neuropsychiatric disorders . The lack of any systematic drift either in the fa or md values along with the relatively small variances suggests the stability of the scanner over the period of time the data was acquired for this study . As reported elsewhere the possible sources of the observed variance are the temperature fluctuations and scanner table vibrations (hunsche et al ., 2001; chenevert et al ., overall, many of our dti findings such as increased md in mtbi in wm structures are in agreement with previous studies (yuh et al ., 2013; inglese et al ., 2005; lipton et al ., 2008; miles et al ., 2008; rutgers et al ., 2008; kraus et al ., 2007; lo et al ., however, it should be pointed out that strict comparison of our dti results with other similar studies is somewhat difficult due to differences in the eligibility criteria for subject recruitment, study population, sample size, and choice of post - injury scan periods . Patients with brain lesions on ct performed within 24 h post - injury were excluded in our study, whereas this approach varied across other recent studies (hulkower et al ., 2013; lo et al ., 2009; kim et al ., 2013). Incidental by the senior neuroradiologist who participated in this study were also excluded . In those cases the lesion appearance did not change from the initial to the follow - up mri . Instead of imaging uninjured subjects for comparison, we recruited patients who suffered orthopedic injury as controls who had similar chronicity of injury and demographic characteristics as the mtbi group . Recruitment of oi subjects accounts, to some extent, for the risk factors that predispose to injury and for the traumatic stress experienced by the mtbi subjects (levin et al ., 2013). Although the criterion for gcs score was 1315, patients who met all of our eligibility criteria and consented to participate had gcs scores of 15, with the exception of two who had a score of 14, indicating normal consciousness or confusion when the patients were evaluated in the ed . Our mtbi cohort had less severe injuries than in other mtbi studies (see for example, kou et al . As described in the results, except for md in certain structures in the right hemisphere, none of the other mri - based measures survived multiple comparison correction based on fwe . The fwe correction is known to be conservative and could lead to type ii errors (false negatives). Another commonly used and less conservative approach is the false discovery rate (fdr; chumbley, 2009). Therefore, we presented our results without the fwe correction when the correction led to results that are statistically not significant . We observed significant differences in md between mtbi and oi at the initial scan that were confined to the right hemisphere . Possible hemispheric asymmetry based on different mri measures was also indicated by others (ling et al ., 2013; niogi et al ., 2008, 2008a; koerte et al ., 2012, 2012a). One possibility for this asymmetry is that the left hemisphere has more densely packed axon branching (klingberg et al ., 1999), presumably making it more resistant to shock - induced damage . However, caution should be exercised in interpreting the finding of the asymmetric increases in diffusivity in the right hemisphere regions . We did not observe any significant differences in the mtr values between mtbi and oi subjects either at the initial or follow - up scans . In one of the earliest mtr studies on mtbi reduced mtr in the splenium of the cc was reported (mcgowan, 2000). However, that study was based on a small sample size with unspecified post - injury scan periods . Whole brain reduction in mtr was reported earlier (hofman et al ., 2002). However, these authors included both moderate and mtbi subjects in their cohort with the gcs in the range of 915 . The post injury scan period varied from 1 to 12 years . From their studies it is unclear if mtbi is associated with reduced mtr in the acute or subacute phases . Quantifying magnetization transfer by a simple measure like mtr limits the potentially useful information about the pathophysiological processes (see for example, garcia et al ., 2012). In contrast, quantitative magnetization transfer (qmt) measures such as concentration of macromolecular pool and exchange rates between the pools could provide more robust information about pathology . Another method that appears to hold some promise is the macromolecular proton fraction (mpf) that is based on mtr effect (yarnykh et al . In fact, this technique was applied to blast mtbi subjects to probe the white matter integrity with promising results (petrie et al ., 2014). Unfortunately we did not acquire the necessary data for qmt and mpf analyses in the current study . Atrophy is thought to represent neurodegeneration that is the result of axonal / myelin loss . Given the mild and uncomplicated nature of injury and relatively short duration of 3 months for follow - up scans, lack of either cross - sectional or longitudinal changes in global and regional volumes in our mtbi cohort is not very surprising . Reduced cerebral volume with time does not necessarily mean atrophy . For example, reduced cerebral volume is expected with the resolution of edema that may be present in the acute phase of injury . The fact that we did not observe any volume changes at the initial or follow - up scan in mtbi relative to oi suggests that significant edema was not present in our cohort . It is difficult to compare our results on atrophy with the published results because of differences in the mtbi and control cohorts, methodology, and post - injury scan periods . For example, atrophy was reported in mtbi after 6 months post - injury (hofman et al ., 2001). In another study on 19 mtbi subjects, atrophy was observed in 4 patients at 37 months post - injury (ross et al ., 2012). In a comprehensive study on atrophy in mtbi global and regional atrophy in the anterior cingulate, left cingulate gyrus, istmus, and precuneus were reported at 1 year after the injury (zhou et al ., 2013). It is possible that we would have observed atrophy in our cohort if we scanned them over longer post - injury time period . Our mrsi analysis showed differences in naa / cr and mi / cr ratios between the mtbi and oi subjects at the initial scan and between the initial and follow - up scans within the mtbi cohort . However, these differences did not survive multiple comparison correction, indicating that any changes in the metabolite concentrations are very subtle . Most of the published studies on mtbi that reported metabolic changes in the acute phase were performed after post - injury day 3 . It is possible that metabolic changes may not be apparent until day 3 post - injury (vagnozzi et al ., 2013). It is also likely that biochemical changes could have disappeared at the 90 day follow - up scan (vagnozzi et al . The results of the previously published mrs studies in mtbi are not always consistent . While some studies found decreased naa (vagnozzi et al ., 2008; henry et al ., 2011; govindaraju et al ., 2004; cimatti, 2006;, 2010; henry et al ., 2010; johnson et al ., 2012) in regions such as parietal lobe, motor cortex, prefrontal cortex, and cc, other studies did not report any changes in naa (maugans et al . Some of these studies used single voxel mrs and this difference in methods could be a contributing factor for the discrepant results . In one report where similar 2dcsi method was used as in our study, no change was found in naa concentration, but cr concentration was found to increase in wm in the mtbi patients (vagnozzi et al ., 2010). Another study using single voxel spectroscopy also detected increased cr level in white matter (gasparovic et al . Since all the above studies that reported decreased naa were based on the naa / cr ratio, including the trends observed in our study, and since increased cr was found in mtbi, it is not clear that decreased naa / cr necessarily implies reduced naa . Moreover, changes in the metabolite levels vary greatly depending on the brain area (gardner et al ., 2014; govindaraju et al ., in addition, the differences in the patient cohort, use of healthy controls, and different post - injury scan times could have contributed to the discrepant results that were reported in the literature . A problem with most of the published mri studies in mtbi is the lack of standardization in patient selection criteria, post - injury scan times, methodology, and control subjects . Classification of tbi as mild, moderate, or severe, based on gcs score which is global and relatively insensitive to mtbi, fails to incorporate newer insights and findings from neuroimaging (manley and maas, 2013). Acquiring data at multiple points that span longer post - injury periods could have provided a better trajectory of pathologic changes, particularly chronic effects . In this study however, this requires the acquisition of unsuppressed water data that was not part of our protocol . While the te used for acquiring mrsi was 53 ms, the default basis set of 35 ms was used for the lc model analysis . While naa, cr, cho, and ins are expected to be less sensitive to the te of the basis set, glx with its relatively short te and j - coupling coupling is more sensitive to the choice of the basis set . This results in poor fitting and relatively large sd . In most cases, both mtbi and oi subjects had received / been prescribed pain medications during their ed visit and which might be in their system for the initial mri . We have not considered the effect of medication on the mri measures . To the best of our knowledge there are no studies that link pain medication and brain mri measures . Finally, we did not attempt to combine all the mri measures for improved detection of neural changes . A major problem with combining the multi - modal data is assigning appropriate weight for each measure.
In the last 20 years, medical informatics has attracted increasing attention and its popularity and has grown considerably (1). This growth has facilitated biomedical research, which has the aim of developing knowledge and using it in the diagnosis, prevention, and treatment of diseases (2). The result of such growth is an unprecedented increase of information in the medical sciences and the creation of millions of databases for storing, organizing, managing, and presenting information to users . One of the most important databases is the medline database which is made available through different hosts . For example, more than 50 sites provided access to medline in 2003, the most important of which were gratfullmed, infotrieve, dimdi, and biomed net (3). Some other hosts also provide medline by subscription, and users who pay for the subscriptions receive more services than those who do not pay . Among these hosts are ovid and elton b. stephens co. (ebsco) host . At present, medline, biomed, and pubmed, which are systems that provide access to medline, have become a necessary part of researchers efforts, and they are being used increasingly by physicians and patients as necessary tools for answering clinical questions (4). However, an important question concerns the extent to which a database, such as medline, has fulfilled users expectations in its ability to reply to clinical questions . Some time ago, medline, which is extraordinarily popular among researchers, included more than 19 million records from almost 5000 publications . However, in recent years, it has expanded such that it had 26 million records in january 2011 that include biomedical and health information starting in 1950 and going up to the present time, and it has been used for more than 2 million searches (4). It can be claimed that it has reliable and documented sources in almost all medical fields, but questions have arisen concerning 1) whether the existing search methods provided by different hosts of medline really direct users to their desired sources and 2) whether the success rate and the rate of acceptance of the information provided are comparable among the different search methods . Looking at each one of these host sites and databases of medline indicates that the different search methods use different search techniques and equipment . Thus, the quality of the searches, with their different strategies, specifications, and updating distance, are different for each site, and researchers must try to decide which methods can provide more accurate information in the shortest period of time (3). This problem is also accompanied by a more important concern, i.e., what methods and strategies provide better help and provide better and more accurate information as the volume of information continues to increase . Thus, users have the difficult decision of choosing among the large variety of services provided by these sites and hosts, especially when special hosts, such as ovid and ebsco, provide a lot of information quickly . For example, ebsco provides four different search methods, i.e., simple search, advanced search and medical subject heading (mesh), and smart search in the medline database and it has added the visual method to this database in recent years by adding clustering algorithms in information retrieval . The important question that researchers must consider is which of these methods and strategies is the best and the most accurate . Each of the methods has advantages and disadvantages and has special characteristics from the user s viewpoint . Perhaps, one cannot definitely determine whether one of these methods is preferred over the others and can fulfill the needs of users completely . However, some of the methods are more popular among users due to the types of algorithms that have been designed based on their own special needs and characteristics . There are two classic criteria that are considered in such an evaluation, i.e., recall and precision . Recall means the ratio of the number of retrieved results relating to subject of search to documents relating to the subject in the entire database, and, generally, it is regarded as the success rate of information retrieval, which cannot be evaluated in the real world of databases due to impossibility of determining all documents relating to a given subject in a database . Precision also refers to the ratio of the number of the related search results to the total retrieved results, and it shows the acceptability of the evaluated documents and is measurable considering special conditions . The determination of precision can clarify the efficiency and effectiveness of these methods if it is determined by real users of information and their judgments at the time they were searching for real information they needed . The use of the mesh - controlled vocabulary search method is available in the medline database and in all of its hosts, and it has been used by researchers for years . The researchers in this research decided to compare these two methods to determine which was more precise from the viewpoint of real users of medline . One of the methods is based on the control of words and terms, and the other is based on the classification and clustering method in terms of precision classic criterion . Of course, the researchers made calculations to assess the two methods and also determined the three types of precision for each method based on the best precision, useful precision, and objective precision formulas, and the two methods were compared on the basis of these results . The results of this research also clarified the comparative efficiencies of these methods for designers of different search methods in databases, and they also can help users choose the search methods that provide the best results for their specific needs . Based on their searches, the researchers concluded that the precision of the search methods in the medical databases had not been evaluated to date based on the three types of precision, i.e., best precision, useful precision, and objective precision, which makes the results of this research more valuable . The following hypotheses were examined in this study: 1- best precision of the retrieved sources in the medline medical database (in the ebsco host) in the visual search method is different from that of the mesh - controlled method.2- useful precision of the retrieved sources in the medline medical database (in the ebsco host) in the visual search method is different from that of the mesh - controlled method.3- objective precision of the retrieved sources in the medline medical database (in the ebsco host) in the visual search method is different from that of the mesh - controlled method . 1- best precision of the retrieved sources in the medline medical database (in the ebsco host) in the visual search method is different from that of the mesh - controlled method . 2- useful precision of the retrieved sources in the medline medical database (in the ebsco host) in the visual search method is different from that of the mesh - controlled method . 3- objective precision of the retrieved sources in the medline medical database (in the ebsco host) in the visual search method is different from that of the mesh - controlled method . This research was conducted using a semi - empirical method, and two search methods were compared, i.e., the visual search method and the mesh - controlled method using ebsco as the host . Considering the precision that the researchers expected for any method and using the ratio comparison formula for both groups (5), a sample size of 287 was obtained for each method . To collect the information, first, 12 training workshops were held for students of higher education in three different majors, i.e., 1) nursing (intensive care nursing, geriatric nursing, and medical / surgical nursing), 2) health information technology, and 3) microbiology and parasitology . The participants were taught the two search methods of medline . To prevent bias towards the search results of one of the methods, the trainees changed the training order of the search methods in the different workshops and taught both methods in some cases . After training, the students searched using both methods in the desired fields and judged the title, abstract, and full text of the article (if available) in the first 20 findings . They even considered whether links were available in the articles to other related articles and scored them based on the relevance of the articles to the subject of interest, using the related checklists in table 1 . The users judged 300 searches for each method, and a total of 7101 titles of articles and abstracts and 3232 full texts of articles were studied and scored by the students . The reason for studying only the first 20 findings was that the users usually tended to review a small part of the findings of each search (between the first 10 to 20 findings), which were referred to in different research reports, such as those of gwizdka and chignell (6). At the end, researchers were asked to express their view about the methods . After collecting the students checklists, scores were determined for each search, and then the precision of each search was determined using the best precision, useful precision, and objective precision formulas (table 2). These formulas were taken from the work of gwizdka and chignell (6), which was based on the model proposed by salampasis, tait, and bloor (7). According to gwizdka and chignell sprecision formulas, in the first formula (best precision), the numerator is the number of articles were awarded a score of 3 in every search (table 1). In the second formula (useful precision), the numerator is the number of articles that were awarded a score of 2 in every search (table 1). In the third formula (objective precision), the numerator is the number of articles that were awarded a score of 1 in every search . The denominator in all three formulas was 20because the first 20 searches were evaluated in every search . For the mean precision in all three formulas, the numerator is sum of the precisions obtained in the two methods, and the denominator is 300(the total number of searches in both methods). The mean average precision (map) for the 300 searches was calculated for both methods . To answer the research hypotheses, the results were entered into spss 11.5 software and analyzed using the independent sample t - test . The opinions of the people who conducted the searches about each method also were studied . The comparison of the averages of best precision in the visual search method and in the mesh - controlled search method showed that best precision of visual search method was greater than that of the mesh - controlled search method (0.1388 versus 0.1142). Statistical analysis of results using the independent sample t testate the 99% confidence level showed that the mean precision of the first method was significantly different from that of the second method (p <0.001). The mean precision averages (maps) of both methods are shown in table 3 . Our research findings indicated that the mean average of useful precision in the visual search method was equal to 0.3758, and, for the mesh - controlled search method, the value was 0.3313 . The independent sample t test in the confidence level of 99% showed that there was a significant difference in useful precision between these two methods (p <0.001). Table 3 gives the mean average of useful precision and the mean precision percent for both methods . The comparison of the averages of objective precision of both search methods showed that the mean precision of the visual method (0.6040) was greater than that of the mesh - controlled method (0.5380). The comparison of these two means indicated that there was a significant difference between the means of the precisions of the two methods (p <0.001)(table 3). Generally, the research findings indicated that60% of the researchers preferred the precision of the visual method of information retrieval for all three types of precision over the precision of the mesh - controlled method . However, 65% of the researchers indicated that the control of keywords in the mesh - controlled vocabulary search method gave them a clearer view about the keywords that should be used for conducting searches, so they were able to select more suitable keywords . Fifty - three percent of the researchers indicated that the use of a combination of the mesh - controlled vocabulary search method and the visual method provided better results . Studies conducted by the researchers did nt reach any conclusion for finding the studies that were conducted with the present research method in the medical databases and measured three types of precision in each search . It can be said that the present research is different from other research efforts in this field from two perspectives: first, many studies have been conducted by specialists using fully predetermined subjects and questions to determine the precision of the search methods, such as the research efforts that were referenced later, but, in the present research, the participants were ordinary users of databases, i.e., students, and the subjects they searched were interesting to them or were based on their need for information . However, the traditional formula used to determine precision was used, but it has some problems . In the present research, the researchers used best precision, useful precision, and objective precision formulas that were presented by gwizdka and chignell (6) and applied by them to study the precision of two search engines, i.e., hotbot, and altavista . Ozel also designed a new kind of search method based on metadata, and he compared best precision, useful precision, and objective precision of these two methods (8). For this reason, researchers have relied on making comparisons of the results of their research with the results obtained by other researchers who measured the precision rate using the traditional technique . In this research the first search method used the medical subject heading of mesh combined with vocabulary, and the second method was the visual method, which is a cluster search method in which searching can be done by combining the keywords of users and the user interface - proposed clusters of ebsco . Findings of the present research confirmed all of the three research hypotheses, and, as the researchers expected, the visual method in ebsco medline had significantly greater precision than the mesh - controlled search method . The mean of the objective precision obtained in the mesh - controlled method was 53.8%, which was in good agreement with the results of previous researchers in this field, i.e., poulter, rubin, and altman(9), who showed precision rates of 44% and 50% in the cut - off points of 10 and 5 times, while the results of research conducted by saka et al . (10) showed that the precision of search in pubmed, one of the free versions of medline that uses the mesh - controlled search method, was 68%, while it was 70% in the ordinary (natural language) search method . (10) showed that there was no significant difference between the precision rates of the two methods they studied . Research by zohour, asadi garakani, and sarabi (2003) showed that the use of the natural method was more precise than the mesh - controlled method . Research by hersh & hickam (11), which was conducted to study the precision and recall of the sources searched in medline using three search methods, i.e., the mesh - controlled search method, textual indexing, and indexing based on words and events showed that textual method was more effective than the other two methods . (12) obtained such research results for the mesh - controlled method was that a lot of the conceptual information presented in abstracts was ignored by the indexers in the mesh method; although a subject heading of the mesh method may be perfect, it cannot be expected to present all of the concepts of interest to the potential users . (10) also referred to these problems and expressed the belief that authors should try to use suitable keywords for their articles, use standard terms, and prepare their abstracts more precisely . It was concluded in all of these efforts that have been referenced that the mesh indexing process should be improved so that it could perform better, because many keywords used in the mesh - controlled method and for searching do nt fulfill all of the users needs . Of course, this does not mean that the use of the mesh vocabulary is not suitable for searching or that searchers should not use it, because each search method has its own advantages . For example, in the present research, 65% of researchers believed that the use of mesh helped them have a clearer understanding of the subject of the search so they were able to use more suitable keywords for their searches . Thus, it can be concluded that, in most cases, the use of combined methods, rather than a special search method, can give better results . This research showed that visual method in ebsco host had better performance than the mesh - controlled method . Then findings of this research are important because, unlike the traditional method, three types of precision were measured, i.e., best precision, useful precision, and objective precision, for the first time in a medical database from the perspective of real users . This new method can provide an improved approach for the evaluation of methods that are used to search databases . Future research should focus on comparing this type of precision with different searching methods available in medline database or other medical databases . The results of such research can lead to the improvement of the evaluation of the methods used to search databases, and they can help designers produce better, more appropriate search algorithms.
A 47-year - old lady presented to us for evaluation of persistent hydroureteronephrosis on imaging . 3 months earlier, she had been treated at another center for a 3 month history of right colicky flank pain associated with malaise and fever, without any lower urinary tract symptoms . Investigations at that time showed raised creatinine (4.8 mg / dl) and bilateral hydroureteronephrosis on ultrasonography . She was treated with antibiotics for suspected pyelonephritis and serum creatinine showed a downward trend, reaching a nadir creatinine of 1.41 mg / dl over the next 3 months . Her medical history was significant for diabetes for 8 years, hypothyroidism for 4 years and hypertension for 3 years . Evaluation at our centre revealed 2 - 4 red blood cells and 6 - 8 white blood cells in the urine . Computed tomography (ct urography) showed bilateral hydroureteronephrosis with global caliectasis, right and left ureter were dilated till s1 and l5 vertebral level respectively, at which point there was a hyperdense enhancing lesion within the ureteric lumen, associated with ureteric wall thickening, periureteric and retroperitoneal fat stranding and normal ureteric caliber below this level . She was planned for cystoscopy and bilateral retrograde pyelography but required emergency bilateral percutaneous nephrostomy for sudden onset anuria, breathlessness, azotemia (serum creatinine 11.1 mg / dl), hypertensive crisis (blood pressure 210/110) and flash pulmonary edema . She had post obstructive diuresis and the creatinine level reached a nadir of 1.53 mg% at 2 weeks . Cystoscopy and retrograde pyelography revealed bilateral mid - ureteric narrowing [figure 2], which persisted on balloon dilatation . A ureteroscope could be negotiated till the lower level of narrowing on both sides and revealed mildly edematous urothelium; no obvious luminal lesion was identified . Intramural pathology compressing the ureteric lumen symmetrically on both sides was considered, and she was taken up for laparotomy and frozen section histology . Venous phase of contrast - enhanced computed tomography of abdomen and pelvis showing: (a) bilateral global dilatation of calyces and pelvis, (b and c) red and blue arrows showing right and left ureters respectively, (d) showing normal urinary bladder retrograde pyelography done before ureteroscopy: (a and b) showing filling defect in right ureter at level of upper sacroiliac joint, both proximal and distal ureter are well opacified with radio contrast, (c) showing filling defect in left ureter, faint opacification of proximal ureter is seen at upper sacroiliac joint on laparotomy, the left ureter was found to be thickened and fibrotic from l5 to s1 vertebral level; similarly, right ureter was thickened and necrotic from l5 to s3 level, retroperitoneal tissue around these ureters was grossly normal . Frozen sections from the ureteric wall on both sides were reported as caseating granulomatous inflammation and later confirmed on routine histopathology . An ileal segment was used to replace the involved ureteric segments from l3 level to the bladder and she was started on 4 drug antitubercular therapy (att), with which she showed clinical improvement (absence of fever, malaise and flank pain). Six months later, creatinine was 0.66 mg% and follow up ultrasound of abdomen 6 months and 18 months post surgery showed resolution of bilateral hydronephrosis . She was continued on att beyond 6 months as evaluation for headache revealed an intracranial right cavernous sinus lesion which was not amenable to biopsy and was presumed to be tubercular in etiology . Bilateral synchronous mid - ureteric lesions are rare and may be congenital, or iatrogenic, following radiotherapy or aortic aneurysmal surgery . Bilateral ureteric involvement with acute renal injury has been described due to radiation - induced mid- and lower ureteric strictures; however, bilateral synchronous mid - ureteric tubercular strictures without obvious radiological renal or bladder involvement as a cause for acute renal injury has not been described in literature . Urinary system involvement in tuberculosis is commonly unilateral, probably due to delayed reactivation of a solitary focus . Ureteric involvement is considered secondary to renal involvement, with the vesicoureteric junction being the most commonly involved . Tuberculosis usually involves the intramural part of the ureteric wall and is characterized by ureteric thickening and post contrast ureteric wall enhancement on contrast - enhanced computed tomography . Bilateral involvement with renal failure is commonly seen after descending infection from one renal unit involving the bladder and subsequent contralateral unit damage due to vesicoureteric reflux and/or small capacity poorly compliant bladder . This case is unique in many respects and cannot be easily explained by accepted theories on the etiopathogenesis of urinary tuberculosis . There was bilateral, synchronous involvement of the mid - ureter, which, according to existing literature, is the least commonly involved ureteric segment in urinary tb . This can only be explained by taking into account the following facts: bilateral pathological involvement of the urinary system via hematogenous route is not unusual; in fact, a seminal study by medlar found bilateral pathological involvement to be the rule, rather than the exception and renal tuberculosis can be clinically silent . Involvement of bilateral ureteric segments either by direct hematogenous spread or following bacilluria from an undetectable renal lesion and subsequent simultaneous activation can explain the findings of the case . The interaction between host immunity and pathogenicity decides the clinical presentation . At one end of spectrum is the activation of a single focus of tuberculosis even in the presence of multiple tubercular pathological foci and at the other end is disseminated tuberculosis where more than one tubercular foci become clinically apparent simultaneously . In the case described above, the simultaneous clinical activation of two pathological foci and later on presentation of an intracranial lesion suggests this patient's immune response lay somewhere midway along the spectrum . The presence of caseating necrotizing granulomatous inflammation is almost always associated with tuberculosis and rarely with nocardiosis (where granuloma formation is rare); however, the clinical features (no solid organ abscesses, response to att) are not consistent with the latter . If tuberculosis had been entertained as a differential diagnosis, appropriate microbiological work up could have been initiated and if proven positive, the patient would have benefited from a 4 to 6 week course of anti - tubercular therapy prior to treatment for ureteric stricture, if it failed to resolve with medical therapy.
Neuroendocrine tumors (nets) comprise a heterogeneous group of neoplasms that are frequently metastatic at the time of diagnosis, and distance of metastatic disease is, next to grading, one of the most important prognostic factors (1)(3). The availability of modern imaging methods for diagnosis and staging of nets has improved at the same time as the spectrum of therapeutic options in the management of metastatic disease has increased during recent years . The frequency of metastatic disease varies depending on the type of tumor, but in specialized centres, 8090% of patients who present with small intestinal nets (carcinoids) and 6070% of patients with pancreatic nets have liver metastases . Histology is the strongest predictor of survival . In the most recent seer (surveillance epidemiology and end results) database analyses, median survival in distant metastatic disease was 33 months in patients with g1/g2 nets and only 5 months in patients with poorly differentiated tumors (neuroendocrine carcinoma (nec) g3) (1). In specialized centres for the treatment of nets, overall 5-year survival rates for patients with stage iv, pancreatic and small intestinal nets are much higher than those reported in the seer database (3). In such centres, 5-year survival rates in patients with metastatic midgut nets exceed 50% (4),(5). In a multivariate analysis of patients with well - differentiated tumors and moderately differentiated nets from the seer database, disease stage, primary tumor site, histologic grade, sex, race, age and year at diagnosis were predictors of outcome (p<0.001). In my centre, in a multivariate analysis of 354 patients with pancreatic nets, the prognostic factors were tnm stage, world health organization (who) classification, ki67, and radical surgery (6). Liver tumor burden, or number of metastases, tumor slope, extrahepatic disease, co - morbidities and performance status represent additional prognostic parameters (3),(7). Retrospective data indicate that circulating chromogranin a (cga) is of prognostic value; highly elevated levels were associated with limited survival (8),(9). Other prognostic markers that are available (e.g., ck19, pten, tsc-2 expression in tumor tissue) require further validation (10),(11). The initial diagnostic approach in patients with nets includes histological examination, which is always required before therapeutic decisions are made . Clinicians should also consider performing repetitive biopsies to reassess the prognosis if the disease course changes significantly . The following investigations are also required: (a) immunohistochemical markers and detailed histological analysis; (b) assessment of the primary tumor and the extent of extrahepatic spread by imaging, including patterns of hepatic metastases; and (c) biochemical assessment of functionality and general tumor markers . The neuroendocrine signature of a cell is defined by the expression of general and specific neuroendocrine markers . General neuroendocrine markers are observed in all cell types, and include the cytosol antigens neuron - specific enolase (nse) and protein gene product 9.5 (pgp 9.5), as well as the secretory vesicle antigens of chromogranin family a for large dense core vesicle (ldcv) and synaptophysin for small synaptic vesicles (ssv). Other neuroendocrine tissue markers are the atp - dependent vesicular monoamine transporter isoforms (vmat1 and vmat2), neuroendocrine secretory protein 55 (nesp55), synaptic vesicle protein 2 (sv2) in both ldcvs and ssv, and neural cell adhesion molecule (n - cam) (12),(13). Immunohistochemical determination of cga and synaptophysin as well as proliferation marker ki-67 (mib-1) is mandatory . In patients with multiple endocrine neoplasia type 1, specific markers such as gastrin, insulin, and pancreatic polypeptide (pp) should be determined . For patients with unknown primary tumors, ttf1 (bronchial / lung), cdx2 (intestinal serotonin - midgut), and pp / islet-1/glucagon (pancreatic) the current who 2010 classification introduces the definition neoplasm to encompass low- to high - grade neuroendocrine tumors (16). At variance with the who 2000 classification, the neuroendocrine connotation is enforced, in recognition of the expression of antigens shared with nerve elements . The classification itself uses a common definition frame that is based on grading and specific staging tools . The definitions are net for the previous carcinoid/well - differentiated endocrine tumor / carcinoma, and neuroendocrine carcinoma (nec) for the previous small cell / poorly differentiated carcinoma . Staging is performed with the familiar tumor node metastasis (tnm) approach according to the anatomical location of the tumors, and this approach is recommended by the who, the american joint committee on cancer and the international union against cancer . However, at variance with the european neuroendocrine tumor society proposal, the who - approved tnm staging system is conceived for carcinoid only, and some parameters for the appendix and the pancreas are different (see table 1) (17)(20). Grading is performed by definition of proliferation using both the mitotic count and the ki-67 index, as proposed by the european neuroendocrine tumor society . Notably, both the who and the american joint committee on cancer endorse such a grading system . A standard computed tomographic (ct) scan of the chest, abdomen, and pelvis or magnetic resonance image is mandatory, and should be complemented by somatostatin receptor scintigraphy including single photon emission computer tomography (spect)-srs and triphasic ct (21),(22). Positron emission tomographic (pet) scanning using 68ga - somatostatin analogue (68ga - dotatoc - pet / ct) is an alternative if available, as it has higher resolution than additional somatostatin receptor scintigraphy . 68ga - dotatoc - pet / ct may help to identify the primary tumor, and is a reliable method for early detection of bone metastases in patients with nets (23)(25). For the detection of small pancreatic nets, endoscopical ultrasonography seems superior to pet / ct (26). In general, pet should be replaced by pet / ct; 18f - dopa pet / ct and 5-htp - pet / ct are promising diagnostic tools, and may be considered if they are available and if somatostatin receptor imaging is negative (27),(28). However, their use in the standard work - up may not be recommended at this time . Although recent studies indicate a prognostic value of fdg - pet in well - differentiated nets, it is not recommended as a routine imaging method (29). In special situations, for example if liver transplantation is being considered, fdg - pet / ct can be considered for g2 tumors as well as 18f - dopa - pet / ct or 5htp - pet / ct if available . Investigation of the large bowel may be useful, either by means of a colonoscopy plus ileoscopy, or by means of a colon ct with a neutral enema . In a retrospective analysis of 123 patients with net liver metastases of unknown primary, in 47% the primary tumor was localized in the small or large intestine by lower endoscopy (30). If the primary tumor is suspected to be in the small intestine, double balloon enteroscopy or video capsule endoscopy may be performed if available and considered necessary for the therapeutic management . If the ct study of liver metastases is inconclusive, t2-weighted thin - slice dynamic gadolinium - enhanced magnetic resonance imaging, or, if available, contrast - enhanced ultrasonography, should be performed . Magnetic resonance imaging is considered superior to ct in the detection and follow - up of liver metastases, and is a preferable choice in clinical trials (21). Although there are no standardized imaging techniques to reliably measure liver tumor burden, an experienced radiologist can use such reports to estimate the percentage of liver tumor involved . The minimal biochemical work - up for nets includes circulating chromogranin a and assessment of a specific marker to assess functionality, such as urinary 5-hiaa evaluation in carcinoid syndrome . Additional assessment of insulin, c - peptide, (proinsulin), gastrin, pancreatic polypeptide, vasoactive intestinal peptide, glucagon, and calcitonin should be useful, depending on the functional status of the tumor, clinical symptoms, and histological features . Chromogranins are a family of glycoproteins found in many hormone - producing organs, and early on they were discovered to be elevated in the plasma of patients with endocrine tumors . Plasma chromogranin a (cga) has been reported to be a prognostic biomarker in gep - nets, correlating with hepatic tumor burden and with shorter survival . In the setting of radically operated midgut carcinoids, elevation of cga has been reported to be both a diagnostic marker and an early marker of recurrent disease . A decrease in cga levels has been used as a marker of response to treatment in clinical trials, for which biochemical response usually is defined as a 50% reduction of cga (31)(33). The combination of cga levels and levels of n - terminal pro - brain natriuretic peptide (nt - probnp) correlate significantly with carcinoid heart disease (i.e., right - sided heart failure due to tricuspid regurgitation and/or pulmonic stenosis because of valve fibrosis (probably) caused by elevated circulating serotonin) (34). There are, however, several limitations to the use of cga as a biomarker in gep - nets . Treatment with proton - pump inhibitors can cause a secondary increase in cga as a result of the increased gastrin production . Impaired renal function may cause accumulation of the peptide, which also results in falsely elevated levels . Many patients with midgut carcinoids are initially misdiagnosed with irritable bowel syndrome, sometimes several years before the correct diagnosis is made (35). There have been reports of elevated cga in irritable bowel syndrome and in inflammatory bowel disease . Cga is thus not of value as a screening test in the evaluation of unclear diarrhea . There has been impressive progress in the field of biomarkers as well as molecular imaging . However, we still need more sensitive markers for early detection and follow - up . Molecular imaging is in its early stages and has the potential to be a significant tool in the management of patients with nets.
Members of the burkholderia cepacia complex (bcc) are gram - negative bacteria of the -proteobacteria subdivision and include plant, animal, and human pathogens, with a widespread distribution in natural and man - made inhabitats . These bacteria exhibit an extraordinary metabolic versatility, allowing their adaptation to a wide range of environments . Among the bcc bacteria, several strains of potential environmental application have been identified due to their ability to degrade pollutants in water and soils (e.g., crude oils, herbicides, recalcitrant aromatic compounds, and xenobiotics). A summary of burkholderia strains capable of degrading recalcitrant xenobiotics is available at the biodegradative strain database (http://bsd.cme.msu.edu/). Several bcc strains are also able to produce antifungal compounds and to fix atmospheric nitrogen . Recent evidence suggests that members of the burkholderia genus are ancient nitrogen - fixing symbionts of mimosa legumes particularly adapted to acidic infertile soils . Due to the ability of some strains to promote plant growth, bacteria of the bcc have attracted significant commercial interest as biocontrol, bioremediation, and plant - growth promoting agents, mainly due to their ability to colonize the rhizosphere of several crops of economical interest, like corn, maize, rice, pea, and sunflower . However, these bacteria have also emerged as important human pathogens and the risks associated with the agricultural uses of bcc strains remain unclear . There is a general consensus that the large - scale use of organisms of the burkholderia genus is imprudent until more is known about the fate of biocontrol strains after their release in the environment . The pathogenic mechanisms and traits used by these bacteria, the clinical outcomes of infected patients, and the interaction of the introduced biocontrol strains with environmental and clinical strains need further studies . Presently, there is some evidence that the environment can be a reservoir for the acquisition of novel bcc infections . For example, the epidemic b. cenocepacia strain phdc was recovered both from patients suffering from cystic fibrosis (cf) in the mid - atlantic region of usa, as well as from agricultural soils . In the last 30 years, several epidemiological, taxonomic, and molecular biology studies of bcc strains have been carried out by research groups worldwide, mainly due to the ability of these strains to cause chronic infections among cf patients . The disease results from mutations in the cystic fibrosis transmembrane conductance regulator (cftr), a camp - dependent chloride channel, mainly expressed in the apical membrane of epithelial cells . The genetic defect results in multiple organ system impairment, being the respiratory tract the most affected . Chronic pulmonary infections, although caused by a limited number of bacterial species (e.g., pseudomonas aeruginosa, bcc, staphylococcus aureus, haemophilus influenza, and stenotrophomonas maltophilia), remain the leading cause of death of these patients . The large majority of respiratory infections among cf patients are due to p. aeruginosa strains . Compared to this major pathogen, however, bcc infections are particularly feared by cf patients and their caregivers since the clinical outcome is highly variable and unpredictable, ranging from asymptomatic carriage to the cepacia syndrome . Additionally, in the vast majority of cf patients, pulmonary colonization with bcc is associated with a worst prognosis, including an accelerated decline of the patients' clinical status and an increased risk of death . Bcc bacteria are also important pathogens in other compromised patients, as is the case of patients suffering from chronic granulomatous disease (cgd). Cgd is a rare hereditary disease that is caused by mutations in the subunits of the nadph oxidase complex of the phagocytes, resulting in their inability to produce reactive oxygen species . Invasive bcc infections and pneumonia is the second leading cause of death of cgd patients . There are also some reports of bcc infections in immunocompromised patients such as cancer and hiv patients, and also among immunocompetent individuals [8, 9]. In immunocompetent individuals, bcc strains have been isolated in cases of chronic suppurative otitis media, pharyngeal infections, and paediatric neck infections . In recent years, an increasing number of bacteraemia cases caused by bcc among non - cf hospitalized patients have been reported . Most of these patients have comorbidities such as chronic hemodialysis, diabetes mellitus, congestive heart failure, and malignancy . Among these hospitalized non - cf patients, hemodialysis, permanence in intensive care units, use of central venous catheters, indwelling urinary catheters, and endotracheal tubes are now recognized as risk factors contributing for bcc acquisition . The accumulating reports of nosocomial outbreaks caused by bcc led to the recognition of these bacteria as emergent nosocomial pathogens among non - cf patients, in particular among oncology patients . The bcc comprises at least seventeen distinct species, genetically distinct but phenotypically similar [10, 11] (table 1). Strains from all the bcc species have been isolated from cf patients and from the environment, however, their frequency of isolation is uneven . While the majority of the isolates obtained from cf patients belong to the species b. cenocepacia and b. multivorans, the majority of the enviromental isolates belong to the species b. cepacia, b. ambifaria, b. cenocepacia, and b. pyrrocinia . A considerable phenotypic variability has been found for all the bcc species, even within sequential clinical isolates of the same strain . This phenotypic variability difficults the correct identification of bcc strains by diagnostic microbiology laboratories . Several phenotypic and genetic methods have been used for the identification of bcc species, including whole - cell protein profile, fatty acid analysis, and 16s rrna and reca gene restriction and sequencing analysis . However, the genetic methods have proven to be the most effective for the correct identification of bcc strains . Nowadays, the multilocus sequence typing scheme (mlst) is considered the golden standard method for the identification of bcc species . Mlst analysis compares the nucleotide sequence of seven house - keeping genes of bcc and the information obtained for each strain sequence type (st) is stored in a public database (http://pubmlst.org/bcc/), thus allowing its use worlwide . Recently, this method was redesigned in a nested - pcr mlst format that can be used for the accurate identification of bcc strains directly from sputum samples . This approach allowed the identification of bcc strains in 23 sputum samples obtained from 17 cf patients, of which 8 samples where culture was negative . In addition, the performance of mlst directly with sputum samples also allowed the identification of bcc strains from cf patients with mixed bcc infections or co - infected with p. aeruginosa strains, without the need for strain isolation . Bcc bacteria emerged as important cf pathogens during the 1980s, when some infected patients exhibited a rapid clinical deterioration due to necrotizing pneumonia and sepsis, resulting in early death . This fatal decline in the patient's clinical condition became known as the cepacia syndrome and was not observed for patients infected with any of the other cf pathogens . The key determinants associated with the cepacia syndrome are not completely understood, and both bacterial and host factors are thought to play important roles in determining this dramatic clinical outcome [23, 24]. Several strains of the species b. multivorans, b. cenocepacia, b. cepacia, and b. dolosa have been shown to be highly transmissible among cf patients through social contact [25, 26]. In particular, highly epidemic lineages of the b. cenocepacia species have been described, including the electrophoretic type 12 (et12), the philadelphia - district of columbia (phdc), and the midwest epidemic lineages [27, 28]. These epidemic strains can have an international impact, as is the case of the highly transmissible et12 lineage . This epidemic lineage spread among individuals with cf from canada, uk, and other european countries, being able to replace b. multivorans and causing a high mortality due to its ability to cause the cepacia syndrome [14, 23, 29]. Due to the easy transmission of highly virulent strains among cf patients, segregation measures of bcc - infected patients have been successfully implemented and led to the reduction of the transmission of bcc strains . The prevalence of bcc species varies geographically, being b. cenocepacia the most predominant species in cf centers in north america, while b. multivorans is the most common species in european cf centers . For instance, in the major portuguese cf centre, b. cepacia is the most prevalent bcc species . In addition, an outbreak of b. cepacia was reported and associated with the use of nonsterile saline solutions for nasal application . Bcc outbreaks among non - cf populations, mainly due to strains of the species b. cenocepacia, b. cepacia, and b. multivorans are also well documented . Accumulating evidence points out contaminated pharmaceuticals, cosmetics, disinfectants, and preservative products as major sources of bcc bacteria [9, 34]. This is due to their ability to survive in these products . In hospital settings, these pathogens have been recovered from tap and distilled water, dialysis machines, nebulisers, catheters, blood gas analysers, thermometers, ventilator temperature sensors, solutions, and intravenous fluids . One of the major problems associated with bcc infection is their intrinsic resistance to most of the clinically available antimicrobials, including aminoglycosides, quinolones, polymyxins, and -lactams . The multiresistance of bcc bacteria appears to result from various efflux pumps that efficiently remove antibiotics from the cell, decreased contact of antibiotics with the bacterial cell surface due to their ability to form biofilms, and changes in the cell envelope that reduce the permeability of the membrane to the antibiotic . Bcc bacteria are also resistant to neutrophil - mediated non - oxidative killing and to the antimicrobial peptides produced by airway epithelial cells, including lysozyme, lactoferrin, and phospholipase a2 . Therefore, cf patients chronically infected with bcc are difficult to treat and, although current treatment strategies use double or triple antibiotic combinations to achieve bactericidal activity, they rarely result in the eradication of the pathogen, particularly in the case of chronic infection . In 2003, the wellcome trust sanger institute sequenced the first genome of a bcc strain . The strain chosen was the type strain of the et12 epidemic lineage, the b. cenocepacia strain j2315 (http://www.sanger.ac.uk/projects/b_cenocepacia/). Presently, the genomic sequences of 18 strains from 7 bcc species are publicly available (http://pathema.jcvi.org/cgi-bin/burkholderia/pathemahomepage.cgi). The genomes of bcc bacteria are organized in three circular chromosomal replicons and one to five megaplasmids, ranging from 6.2 to 8.7 mbp in size, with a gc content of about 67% . The large size and repartition of the genomes of bcc is thought to increase their flexibility to acquire and lose genes . In a recent bioinformatics study, (2010) suggested that the genes located in secondary chromosomes exhibit a weaker codon usage bias than those located in primary chromosomes, being subject to a faster evolutionary rate . Several evidences point out that more than 10% of the bcc genomes have been acquired by horizontal gene transfer, contributing to the genomic plasticity and metabolic diversity of these bacteria . For example, in the case of the b. cenocepacia strain j2315, 14 genomic islands, most probably arisen from horizontal gene transfer, have been identified based on their distinct gc content percentage . The acquisition of genomic islands appears to play a crucial role in the evolution of this particular epidemic lineage, introducing new functions that promoted survival and pathogenesis in the cf lung . This is the case of the 31.7 kb cci pathogenicity island, which appears to be unique to b. cenocepacia strains . This pathogenicity island encodes both virulence and metabolism - associated genes, including the cciir quorum sensing system, a fatty acid biosynthesis operon, transcriptional regulators, and genes involved in the metabolism of amino acids . In addition, the genome of b. cenocepacia j2315 contains 79 insertion sequence (is) elements that are most probably involved in genomic rearrangements, replicon fusion, activation / silencing of gene expression, mobilization of dna, and recruitment of foreign genes . Another feature of the genomes of bcc bacteria is the presence of multiple pathways with related functions, and gene redundancy due to the occurrence of paralogous genes . Sequencing of several bcc genomes, followed by comparative genomics, is a powerful tool for the identification of virulence - associated genes of bcc bacteria, including new genes encoding proteins with no predicted function . In the sequenced bcc genomes, the percentage of protein encoding genes with unknown function varies between 13 and 35% (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi). It is quite possible that a significant percentage of these genes of unknown function might be involved, either directly or indirectly, in the pathogenesis of bcc bacteria . Different strategies have been designed to identify pathogenicity - related genes from bcc bacteria, including the generation of mutant libraries with transposons and plasposons, systematic gene - by - gene inactivation and high - throughput sequencing, as illustrated in figure 1 . Our research group has been using a strategy based on the generation of mutant libraries from b. cenocepacia and b. cepacia strains by random mutagenesis with plasposons, followed by rescue of the interrupted genes, sequencing and comparison of the nucleotide sequence of the interrupted genes with the available genome sequences of bcc strains, combined with the virulence assessment in the bcc infection models x - cgd mice and/or caenorhabditis elegans . A mutant library derived from b. cepacia ist408 allowed the identification of the bce - i gene cluster that encodes proteins and enzymes involved in the biosynthesis of the exopolysaccharide (eps) cepacian . Cepacian is composed of a branched acetylated heptasaccharide repeat unit with d - glucose, d - rhamnose, d - mannose, d - galactose, and d - glucuronic acid, in the ratio 1: 1: 1: 3: 1, respectively . Several studies have shown that cepacian interfered with the phagocytosis of bacteria by human neutrophils and, inhibits neutrophil chemotaxis, and the production of reactive oxygen species [44, 45]. The ability to produce this eps was also associated with persistence of infection in the balb / c and x - cgd mice models of infection [44, 46]. Studies performed with cepacian - defective mutants have also shown that cepacian is required for the formation of thick and mature biofilms . Biofilm formation in vitro is a common trait of bcc strains and has been associated with the persistence of bcc infections . In addition, bacteria of the bcc growing in biofilms have been found to be more resistant to antimicrobials than those growing plancktonically . It is also worth to mention that in a recent study by dales et al . (2009), biofilms formed by bcc were found to be more resistant to antibiotics compared to p. aeruginosa biofilms . Remarkably, a mutant producing about one half of the amount of the eps was recently found to carry a plasposon insertion interrupting a gene encoding the rna chaperone hfq . Hfq proteins are global regulators of metabolism, acting as rna chaperones involved in the riboregulation of target mrnas by small regulatory noncoding rnas (srnas), facilitating the interaction with their target mrnas . The b. cepacia hfq mutant was shown to be more susceptible to stress conditions, particularly to those that mimicked the lung environment of the cf host, indicating that hfq plays a major role in the survival of bcc bacteria under those stress conditions . In addition, the hfq mutants from b. cepacia, b. dolosa, and b. ambifaria exhibited a reduced ability to colonize and kill the nematode c. elegans, indicating that hfq is an important virulence determinant of bcc bacteria . In agreement with the roles played by hfq in other bacteria, sousa et al . (2010) have also shown that the b. cepacia ist408 hfq is able to bind to srnas . Recently, 213 putative srnas were identified within the genome of b. cenocepacia j2315, based on the combination of comparative genomics and prediction of their secondary structures . Work in progress envisages the identification and characterization of srnas from bcc to gain clues on their possible contributions to virulence . Another mutant library, derived from the highly epidemic strain b. cenocepacia j2315, allowed the identification of a gene encoding an acyl carrier protein (acp). Bacterial acps play a central role in metabolism, being the donors of the acyl moiety that is required for the biosynthesis of fatty acids, phospholipids, endotoxins, glycolipids, and signalling molecules that are necessary for growth and pathogenesis . The acp mutant exhibited an increased ability to form biofilms in vitro, a more hydrophobic cell surface, and reduced ability to colonize and kill the nematode c. elegans, indicating that acp protein is a virulence determinant for bcc bacteria . In addition, the amino acid sequence and structural differences between the acp proteins from bacteria and humans make this protein an attractive target for the development of novel antimicrobial compounds . The screen of this library for mutants impaired in their ability to kill the nematode c. elegans allowed the identification of the regulatory protein pbr . The pbr mutant exhibited a pleiotropic phenotype, being unable to produce phenazines, exhibited a reduced resistance to stresses such as oxidative and osmotic stress, and a reduced ability to survive prolonged nutrient starvation periods . A signature - tagged mutagenesis (stm) strategy was used by hunt et al . To identify putative virulence factors of bcc (figure 1). Stm is a comparative hybridization technique that uses a collection of transposons, each modified by the incorporation of a dna sequence tag . The pool of mutants is inoculated into a chronic pulmonary infection animal model and the bacteria recovered after infection are identified due to the tags . Mutants containing a transposon insertion in genes required for survival will fail to pass through the in vivo selection, thus allowing the identification of these genes . This strategy led to the identification of several b. cenocepacia k56 - 2 genes that were required for bacterial survival in a rat model of chronic lung infection, including genes involved in cellular metabolism, global regulation, dna replication and repair, cell surface proteins, and polysaccharide production . A suppression - subtractive hybridization (ssh) strategy was used to identify genes that are unique to the b. cenocepacia and/or to et12 epidemic lineage strains (figure 1). Recently, a high - throughput sequencing strategy was used to compare the transcriptional response of clinical and environmental strains of b. cenocepacia (figure 1). This strategy revealed a large number of regulatory differences between environmental and clinical strains, which might result from specific adaptations to each of the different niches, despite their high degree of dna sequence similarity . Genes that encode for molecular chaperones and iron acquisition proteins were found to be particularly induced in the clinical strain . All these strategies have allowed the identification of several genes putatively involved in the virulence of bcc strains . However, the characterization of knockout mutants in these genes is hampered by limited available genetic tools and the inherent resistance of bcc strains to the most common antibiotics used for genetic selection . In this context for instance, lefebre and valvano (2002) constructed several expression vectors that contain the dhfr gene, encoding the dihydrofolate reductase enzyme required for trimethoprim resistance, together with either the constitutive promoter of the s7 ribossomal protein gene from burkholderia sp lb400, or the arabinose - inducible bad promoter from escherichia coli . However, the concentration of arabinose required for maximal gene expression [2% (w / v) or higher] causes a change in cell volume typical of osmotic stress . As a consequence, the full complementation of a given mutation using these vectors is seldom achieved, limiting its use . Therefore, those authors have constructed another expression vector, containing the rhamnose - regulated prhab promoter of e. coli that allows maximal gene expression at low concentrations of rhamnose . Another limitation derives from the fact that the b. cenocepacia j2315 strain is a poor recipient of dna in transformation and conjugation . As an example, transformation of b. cepacia ist408 with plasposon ptnmod was 10-fold more efficient than b. cenocepacia j2315 (s. a. sousa and j. h. leito, unpublished results). Recently, the electroporation procedure for this strain was modified to increase its transformation efficiency . Factors that contribute to this improvement include the addition of glycine to the growth medium to weaken the thick cell wall, demethylation of transforming dna by extraction from a e. coli dam dcm host strain to escape to the j2315 restriction system specific for methylated gatc sites, the inclusion of the ocr protein in the transformation mixture to act as a decoy to inhibit type i restriction endonuclease attack of entering dna, and the use of spermine to reduce the resistance of the b. cenocepacia j2315 strain to several antibiotics . Other strategies used to effectively generate bcc mutants rely on the lambda red recombinase system, as proposed by datsenko and wanner (2000). This strategy uses linear dna transformations and has allowed the successful homologous expression of a lipase gene in b. cepacia, as well the construction of an insertion mutant in b. cenocepacia j2315 bcal1538 (c. g. ramos, s. a. sousa, j. h. leito, unpublished results). A combination of one or more of the previous approaches has allowed the identification of several potential virulence factors, including the cable pili and various adhesins, flagella, a type iii and a type vi secretion systems [67, 68], lipopolysaccharide, four types of iron - chelating siderophores (salicylic acid, ornibactin, pyochelin, cepaciachelin, and cepabactin), production of extracellular proteins, like proteases, lipases, and haemolysins [71, 72], quorum - sensing systems [73, 74], and others (recently reviewed in). However, not all strains produce each of these virulence factors, and none of these factors has been clearly demonstrated to be a major contributor to human disease . In fact, contrasting with other pathogens, the pathogenicity of bcc bacteria does not rely on a single gene . Accumulating evidences point out that bcc virulence is polygenic, involving genes related to survival under stress conditions [50, 7678]. Nevertheless, the bcc genome is equipped with the known crucial genes for colonization and initiation of chronic infection in the respiratory tract, which are involved in motility, adhesion, and host tissue damage . Another important feature of some bcc strains is their ability to invade and survive inside eukaryotic cells, including soil - dwelling amoebae, human macrophages, and airway epithelial cells . Members of the bcc have emerged in the last decades as important pathogens to human, animals, and plants . The pathogenicity of these bacteria is polygenic, and thus involves a multitude of known and unknown virulence factors and determinants . Several strategies have been successfully used by several research groups to reveal novel and unknown virulence factors and determinants . The knowledge of the molecular mechanisms employed by bcc bacteria for virulence and pathogenesis is of crucial importance to identify new targets for the rational design of novel strategies and/or molecules to combat bcc infections, since their resistance to most of the clinically - relevant antimicrobials renders the infections untreatable . In order to be regarded as a potential drug target, a given gene or gene product must be essential for survival of the pathogen in the host and should be conserved in the various strains of the pathogen, while presenting little or no conservation in humans . Genome - based strategies, including genome sequencing, microarray - based expression technology, and large - scale mutagenesis studies, are expected to contribute, in the near future, for the development of new strategies and/or antimicrobials molecules to fight the devastating and presently difficult - to - treat infections caused by bcc strains.
A 36-year - old man presented with sudden severe occipital headache, and neck stiffness in may 1999 . After several weeks, the headache subsided, however, blurred vision subsequently developed . T1-weighted (665/14/2 [repetition time / echo time / excitation]) and t2-weighted (4200/99/2) mr images showed a large cystic mass with a small solid portion in the left temporal lobe (figs . Gd - dtpa enhanced t1-weighted images showed marked enhancement of a thick peripheral wall and solid portion (fig . 1c). On diffusion - weighted mr images (b value=1000 sec / mm, isotropic image), the mass was surgically excised, and histopathologic examination revealed a typical adamantinous craniopharyngioma with anastomosing epithelial islands and a palisaded layer of cells, as well as an area of keratinization and numerous calcifications (fig . Craniopharyngiomas are generally considered to be epithelial tumors arising from remnants of the craniopharyngeal duct, which connects the stomodeal ectoderm with the evaginated rathke's pouch, which in turn forms the adenohypophysis (7, 8). This theory suggests that craniopharyngiomas can arise anywhere along the migration of rathke's pouch, which extends from the vomer and the roof of the nasopharynx, through the midline sphenoid bone to the floor of the sella turcica . Thus, craniopharyngiomas can potentially arise in unusual locations such as the nasopharynx (2, 8), sphenoid bone (9), third ventricle (3), and posterior fossa (10). However, the above hypothesis does not explain the development of craniopharyngiomas either in the pineal gland (5) or the temporal lobe as in our case . There is no clear embryological reason for craniopharyngiomas to originate from the pineal gland or temporal lobe . (6) suggested that they might originate from totipotential or multipotential cells that reside in the pineal gland . However, metastasis has never been described in this benign neoplasm, and therefore we think that the former hypothesis is more reasonable than the latter . Craniopharyngioma would not usually be included in the differential diagnosis of a temporal lobe mass . Given the well - defined cystic mass with peripheral dense enhancement, our preoperative diagnosis was pilocytic astrocytoma . Craniopharyngiomas can be classified into two histopathologically and clinically distinct subtypes (i.e. Adamantinous and squamous - papillary variants) (8). The adamantinous type consists of a predominantly cystic lobulated tumor, which is often observed in an intrasellar / suprasellar location in children . These cysts contain various amounts of cholesterol, triglycerides, methemoglobin, protein, desquamated epithelium, and watery fluid content . Squamous - papillary craniopharyngioma, on the other hand, consists of a predominantly solid or mixed solid - cystic spherical tumor in a suprasellar location in adults . The solid tumor parts have an inhomogeneous but intense enhancement with small necrotic areas, and calcifications are rare . The combination of papillary and adamantinous tumor parts within the same neoplasm has been described in 15% of these tumors . In summary, although our case did not show any specific radiologic finding permitting the differentiation of craniopharyngioma, to the best of our knowledge, this is the first case of a craniopharyngioma originating in the temporal lobe . It does not appear to be embryologically derived from ectopic embryonic remnants of the craniopharyngeal duct.
A 22-year - old female reported with complaints of multiple missing teeth in upper and lower anterior region . On examination, patients skin was dry and soft on face and increased thickness of nails; palm and sole were normal . The patient had 18, 15, 12, 22, 27, 28, 38, 45, and 48 missing teeth with generalized microdontia . The canines had cone shaped clinical crowns, and molars appeared malformed [figure 1] with obliterated occlusal tables . Vertical dimensions were reduced due to a lack of occlusion and vertical stop causing protuberant lips . The panoramic radiograph confirmed presence of retained primary teeth in both the arches . With the above findings and orthopantomogram (opg) figure, we came to a conclusion and diagnosed as haed . To restore the missing teethto increase the vertical dimensionto correct size of teethalignment of teeth . To restore the missing teeth to increase the vertical dimension to correct size of teeth alignment of teeth . Treatment plan included compensating microdontia by increasing tooth size using fpd retained by implant . To use implants, enough space was required; so orthodontic alignment of teeth was required . As teeth were distributed unevenly, it had to be aligned to gain space for implants . Retained deciduous teeth were extracted and orthodontic correction [figure 2] done with aligning and leveling . Orthopantomograph during orthodontic correction treatment two implants [figure 3] were placed in region of 12, 13 and one in the region of 45 . Final fpd [figure 4] was given and orthopantomograph [figure 5] taken posttreatment . Intraoral photograph posttreatment orthopantomograph posttreatment the prosthetic rehabilitation provides the patient with esthetic, functional, and phonetic benefits, as well as a better outlook towards her social life and peers oral hygiene instructions, were given to the patient, and he was asked to report for follow up and assessment of her fixed prosthetic appliance after 6 months . To restore the missing teethto increase the vertical dimensionto correct size of teethalignment of teeth . To restore the missing teeth to increase the vertical dimension to correct size of teeth alignment of teeth treatment plan included compensating microdontia by increasing tooth size using fpd retained by implant . To use implants, enough space was required; so orthodontic alignment of teeth was required . As teeth were distributed unevenly, it had to be aligned to gain space for implants . Retained deciduous teeth were extracted and orthodontic correction [figure 2] done with aligning and leveling . Orthopantomograph during orthodontic correction treatment two implants [figure 3] were placed in region of 12, 13 and one in the region of 45 . Final fpd [figure 4] was given and orthopantomograph [figure 5] taken posttreatment . Intraoral photograph posttreatment orthopantomograph posttreatment the prosthetic rehabilitation provides the patient with esthetic, functional, and phonetic benefits, as well as a better outlook towards her social life and peers oral hygiene instructions, were given to the patient, and he was asked to report for follow up and assessment of her fixed prosthetic appliance after 6 months . The typical treatment of a patient with ed is achieved in several phases, depending on the craniofacial development, thus requiring a comprehensive and multidisciplinary approach . The different treatment modalities are dictated by manifestation of level of hypodontia and resulting malocclusions . Numerous clinical reports have demonstrated importance of prosthetic dental treatment in ed patients for psychological and psychosocial reasons . A number of reviews regarding treatment of ed has showed that implant therapy in early infancy or adolescent age while little information is present in literature for ed patient above 18 years of age . Prosthodontic treatment of ed can include fixed, removable, or implant supported prosthesis of which implant and fixed partial prosthodontics are the most frequent modalities . In removable prosthesis, frequently the oral mucosa is dry due to a decrease in the number of mucous glands and lesser quantity of saliva which makes retention of removable prosthesis more difficult . Overdentures can be used to restore ideal occlusion and usually allow preservation of existing dentition, but these require rigorous oral hygiene regimes to avoid development of caries and periodontal problems . This case report describes a routine method for fixed prosthodontic treatment of a patient with ed . As the patient was older than 18 years, the growth period was assumed to be over that would have resulted in the failure of fpds as shown by hogeboom . A study by guckes et al ., showed that patients younger than 18 years had a hazard ratio of 2.8 compared to the patients older than 18 years . So for young patients, fpds should be avoided as they could interfere with jaw growth and implants should be recommended and vice versa in patients> 18 years . The dental appearance of patients affected by ed has to be improved for physiologic and psychologic reasons, so that they can lead a normal social life with self - esteem . This clinical report demonstrates the implant retained fpd as a suitable treatment while treating an adult patient with ed . This treatment option gives excellent result, which not only improves masticatory function but also enhances esthetics, allowing patients to lead a normal social life.
Seasonal influenza is a known cause of morbidity and mortality among cancer and transplant patients . During influenza season, 20 to 30% of stem cell transplant sct recipients with respiratory symptoms can test positive for influenza with a mortality rate of up to 28% . Non - transplant cancer patients can also have a high mortality rate of up to 38%, being higher in patients with lung, hematological and colorectal cancer, in patients that develop lower respiratory tract infections, and in patients with other co - morbid conditions . In argentina, seasonal influenza in onco - hematological patients is associated to a 12% incidence of pneumonia and to a 5% of 30-day mortality . In march 2009 a novel influenza a virus, later known as 2009 pandemic influenza a (h1n1), emerged in mexico . The new strain initially spread among travelers to the usa and canada, and subsequently infected people worldwide . Clinical presentations ranged from mild symptoms to severe cases that lead to pneumonia and respiratory failure related deaths . The first cases of pandemic influenza a (h1n1) in argentina were reported in may 2009, in travelers returning from mexico and the usa . From may to december 2009 there were 11931 cases of confirmed influenza a h1n1 in argentina, 617 deaths, and over 90% of the circulating respiratory viruses in adults were the novel influenza a h1n1 . Data from different studies on the impact of this new virus in the adult cancer and sct population are somewhat contradictory . Many studies from different countries were reported 1, 6 16 . In these studies, the incidence of pneumonia ranges from 20 to 52%, while the reported mortality rate ranges from 010% 6, 11, 12, 14, 16 to as high as 2131% 7 10, 15 . During the 2013 winter season, pandemic influenza a h1n1 continued to circulate (flunetdb, who, http://apps.who.int/globalatlas/dataquery/default.asp). In this study, we examined the effects and severity of pandemic h1n1 influenza during the 2009 influenza season, in patients with cancer and sct in two cities of argentina . To august 2009, cancer and sct patients older than 16 years, who presented a confirmed influenza infection by real - time pcr were included . The following data were obtained anonymously: underlying illness, type and date of sct, whether patients were or were not receiving immunosuppressive treatment, at the time of the influenza diseases, immunization for seasonal influenza, clinical presentation (influenza like or pneumonia), laboratory and radiology results, anti - viral treatment, and outcome . In addition, data on the time between the onset of symptoms and the initiation of antiviral therapy, need for ventilation support, and presence of co - infections were also collected . The rt - pcr tests for pandemic influenza a h1n1 virus were performed on nasopharyngeal swabs or bronchoalveolar lavage samples when available . Either of two pcr protocols were used for detection of the pandemic influenza a h1n1 virus depending on test availability: the real - time ready influenzaa / h1n1detectionset version june 2009 (roche diagnostics gmbh, roche applied science68298 mannheim, germany) and the pcr protocol used by the who (cdc protocol of real - time rtpcr for influenza a h1n1 28 april 2009, revision 1, 30 april 2009). Categorical variables are shown as percentages and they are compared with the -distribution test or fisher test . The association between baseline variables and events is presented as or with the 95% ci . In all cases, from may to august 2009, 12 centers sent data of 65 cancer patients with 2009 h1n1 virus disease confirmed by positive pcr in bal (3) or nasopharyngeal wash (62). The median age of the patients was 51 years (range 17 to 81), and 57% were female . The majority of patients (47) had onco - hematological cancer (72%) and 18 (28%) had solid tumors . Cancer treatment included chemotherapy (46), sct (16), no treatment (2) and surgery (1). The median time of patients follow up from the onset of symptoms was 61 days, range 5 to 259 . Pneumonia and pneumonia with oxygen saturation <96% were the most common clinical presentations (43/65, 66% and 30/65, 46%, respectively). Co - infections were present in a minority of cases (9/65, 14%) and only among patients with community acquired influenza . * 4 pneumonias (3 s. pneumoniae, 1 moraxella catarrhalis), 3 bacteremia (k. pneumoniae, mrcns, streptococcus group c); * * influenza b and parainfluenza 3 infection . Patients started treatment at a median of two days from onset of symptoms (range 0 to 45 days). Sixty eight percent (43/63) of patients started treatment within the 48h after the onset of symptoms . Some patients received combined antiviral treatment because of the potential circulation of seasonal influenza a h1n1 known to be resistant to oseltamivir . Most patients acquired the infection in the community (58, 89%) while 7 (11%) of infections were acquired in the hospital setting despite the implementation of adequate standard precautions and isolation measures during this outbreak . Detailed descriptions of the outcome of patients with community acquired (capia) and nosocomially - acquired (napia) pandemic influenza a h1n1 infection patients are described in figure 1 and figure 2 . The 30-day mortality was higher among patients with napia (3/7, 43%) than among those with capia (9/58, 15,5%). Urti: upper respiratory tract infection; icu: intensive care unit; mv: mechanical ventilation; ards: acute respiratory distress syndrome . Urti: upper respiratory tract infection; icu: intensive care unit; mv: mechanical ventilation; ards: acute respiratory distress syndrome . Reasons for patient admission included mainly oxygen desaturation, but, in many cases, patients were admitted because of their severe state of immune suppression and the lack of information about this emergent virus, especially when the patient s social environment prevented him / her from easy access to medical care . Outpatients who presented with upper respiratory tract symptoms (urti), had the most benign course since the majority (11/19, 52%) resolved their infections with antiviral therapy in the outpatient setting, and, among the 8 (42%) who were admitted, none of them required icu admission or developed signs of pneumonia . The 30-day mortality among capia urti was 0 . Outpatients who presented with pneumonia had a more severe course since almost all of them (37/39, 96%) were admitted, 15/39 (38%) required icu, 11/39 (28%) required mechanical ventilation (mv), and the 30-day mortality in this group was of 23% (9/39). The worst prognosis in this group was seen among those who presented with pneumonia and desaturation (25), leading to an admission rate of 100% (52% in icu, 44% needed mv), and a 30-day mortality of 36% . Patients, who developed napia, belonged to 3 different centers and started having symptoms at median of 20 days after admission (range 2 to 33). This group had the poorest prognosis since the 30-day mortality rate was 43% (3/7). One of three (33%) napia urti progressed to pneumonia, while none of the 19 patients with capia urti did . Therefore, the overall progression from urti to pneumonia was of 4.5% (1/22). The 30-day mortality according to the clinical presentation and setting is best described in table 2 for comparison . It is shown that having pneumonia at presentation and developing of the infection in the hospital setting tended to be associated with a higher 30-day mortality without achieving statistical significance . Urti: upper respiratory tract infection; capia: community - acquired pandemic influenza a infection; napia: nosocomially acquired pandemic influenza a infection . Bacterial complications were documented in 6 (9%) patients and included 3 bacteremias (cvc related mrcns, acinetobacter baumanii, and gnr that was only seen in direct examination), 2 pneumonias (mrsa, s. pneumoniae) and 1 meningitis (ps . Aeruginosa). The median time from the onset of symptoms to the development of a bacterial complication was 11 days, range 034 . Bacterial complications developed only among patients who presented with pneumonia (6/43, 14%) by the pandemic influenza a h1n1 . Non - infectious complications developed in 14 (22%) patients . They included: renal failure (5), respiratory failure (5), shock (3), hypokalemia (3), nonbacterial infections (3) (cmv reactivation, candidiasis by c. glabrata, and pcp) and bleeding (2) (lung and brain). No deaths were observed among patients who had been vaccinated against seasonal influenza in the same year . However, we do know that the first case was detected on may 12, while the seasonal influenza vaccine was available since march . Therefore, there is a high probability that at least 14 days might have passed between vaccination and the onset of symptoms . The presence of any co - infection (bacterial or viral) at onset of symptoms and the delay in treatment were not associated to death or mechanical ventilation . By univariate analysis lack of history of vaccination, and the following baseline characteristics: pneumonia, oxygen saturation <96%, and lymphocyte count <800 cells/l, were associated to 30-day mortality and mechanical ventilation . By multivariate analysis only lack of history of vaccination (or did not apply because none died in the vaccinated group) and baseline oxygen saturation <96% (or 19.5; 95% ci 2.28 - 165.9; p=0,007) were associated to mechanical ventilation and death . There might be a bias regarding the apparent benefit of vaccination because in cancer patients, immunization is usually advised when the period of major immunosuppression has finished . Sct: stem cell transplantation; gvhd: graft vs host disease; chemo: chemotherapy; is: immunosuppression; urti: upper respiratory tract infection; nfw: nasopharyngeal washing; bal: bronchi - alveolar lavage; ifd: indirect immunofluorescence assay; chest ct: chest computed tomography; hd: hodgkin disease; all: acute lymphoblastic leukemia; cll: chronic lymphoblastic leukemia; mm: multiple myeloma; aml: acute myeloid leukemia; cml: chronic myeloid leukemia click here for additional data file . Our study shows the clinical course of the infection by the 2009 pandemic influenza a h1n1 virus in 65 cancer patients from 12 institutions located in two cities of argentina . Overall we found a high rate of pneumonia (66%) and mortality (18%). The clinical course was less severe in those who presented with an urti in the outpatient setting in contrast to those who presented with pneumonia and desaturation especially in the hospital setting . We also found that the best predictors of death were oxygen desaturation at presentation and lack of vaccination against seasonal influenza . The incidence of pneumonia we found is higher than the one reported with seasonal influenza in cancer patients (544%) 3, 18 20 and it is also higher than the incidence of lower respiratory tract infections (lrti) caused by the 2009 pandemic influenza a h1n1 in the hospitalized general population (4044%), in solid organ transplant recipients (23%), in hct recipients (2156%) 1, 6, 8, 12 14, 22 and in patients with hematological malignancies (48%). Only one small study that includes 15 confirmed cases of 2009 pandemic influenza a h1n1 infection in onco - hematological patients reports a higher incidence of pneumonia (87%). The 30-day mortality rate we show in this study is more than three times the one observed in argentina in the same patient population when looking at infections by other respiratory viruses such as adenovirus, influenza, parainfluenza, and rsv (5%). However, it is similar to the mortality rate reported in hematological patients with pneumonia by influenza, parainfluenza, picornavirus and rsv at a us institution (15%). It is well known that seasonal influenza - induced pneumonia is independently associated with mortality after hct (adjusted hr 2.6; 95% ci 1.40 - 4.86). The pandemic influenza a h1n1 virus is an independent risk factor for progression to lrti (or 5.64; 95% ci 1.3 - 25) and hypoxemia (or 5.91; 95% 1.424) compared with seasonal influenza virus in hct recipients . In addition, immunosuppression was a main risk factor for early mortality among 337 argentinean patients admitted to icu with influenza like illness and respiratory failure that required mechanical ventilation . These data explain our high mortality rate observed among the 17 patients who were admitted to icu (11/17; 65%) or among the 12 patients who developed respiratory failure (11/12; 92%). These values are comparable to those reported in the same type of population 1, 8, 12, 22, but are higher than those reported in other populations, which ranged from 024% 21, 26 29 . Indeed, the overall mortality rate observed among argentinean patients admitted to icu and requiring mechanical ventilation was 46% . To further support the high mortality of patients with pandemic 2009 influenza pneumonia, we identified hypoxemia at onset of symptoms as an independent predictor of mortality . Lymphocytopenia has been described as a risk factor for progression from upper to lower viral respiratory tract infection in cancer patients 24, 30, and profound lymphopenia (<100 cell/l) was reported as a significant risk factor for requirement of mechanical ventilation and death in hct recipients infected with seasonal influenza virus . In our study, having fewer than 800 lymphocytes/l at presentation was a predictor for the need for mechanical ventilation and death in a univariate but not in a multivariate analysis . We did not analyze a lower value such as <100 of lymphocytes due to the small number of patients included with this value . It is noteworthy that co - infections or bacterial complications developed in less than 15% of patients . Neuraminidase inhibitor therapy appears to be effective in preventing progression to lrti 2, 30 and hypoxemia when instituted early after onset of symptoms . It was reported that delaying therapy in cancer patients with the pandemic influenza a h1n1 virus infection was significantly associated with death . Early initiation of antiviral therapy in these patients may attenuate the severity of disease 21, 27 . In our series, antiviral therapy was started early after a median of two days after the onset of symptoms, with a range from 045 days . We did not find any correlation between days from onset of symptoms to therapy or diagnosis to therapy by univariate or multivariate analysis . It is known that patients with urti can be treated as outpatients and can recover completely from their infection . In our series half of the outpatients with urti remained as such, while the other half was admitted but did not require icu . There is a possibility that most of the admitted patients could have been managed as outpatients as well . The global progression from urti to pneumonia in our study was of 4.5% (1/22). This single patient had a nosocomial infection and died 21 days later with sepsis, respiratory failure and neutropenia . This is according to reports of progression to lrti that may occur even after one week of symptoms . In contrast, patients with lrti required hospitalization with a high number of them requiring admission to icu for ventilation support . The dismal outcome seen in these patients despite treatment with oseltamivir probably indicates that this high - risk group needs to be treated differently from patients with isolated urti . Some authors have suggested an initial treatment with high dose of oseltamivir and/or combination therapy approaches in the case of respiratory failure . All our patients received standard dose of oseltamivir (75 mg po twice a day) for a minimum of 10 days based on data on slower viral clearance . Nosocomial outbreaks of seasonal and pandemic 2009 influenza a h1n1 infection can develop even in the setting of appropriate infection control measures . This mortality rate is higher than the previously reported 1327%, however, the number of patients in our report is too small to make any conclusion . Seasonal influenza vaccination is recommended yearly for all patients with cancer and hsct recipients . In our study all deaths occurred among the non - vaccinated patients, while there were no deaths among the vaccinated patients . Individuals vaccinated against seasonal influenza a or with previous seasonal influenza infection may benefit from preexisting cross - reactive memory cd4 t cells and cd8 t cells reducing their susceptibility to influenza a h1n1 infection or explaining, at least in part, the unexpected mild illness in the community 35 39 . Whether the trivalent seasonal influenza vaccine is protective against the pandemic influenza a h1n1 virus in cancer patients is still a matter of debate . In conclusion, we report a series of cancer patients with the pandemic influenza a h1n1 infection with a high incidence of hospitalization, severe pneumonia, icu admission, mechanical ventilation, and 30-day mortality . In our series hypoxemia and lack of vaccination with seasonal trivalent influenza vaccine a larger study is needed to evaluate the possibility of cross protection with the seasonal influenza vaccination . Baseline characteristics, clinical presentation, treatment and outcome of 65 cancer or sct patients doi: 10.5256/f1000research.5251.d35377
Hepatitis c virus (hcv) infects 175,000,000 people worldwide and is a major cause of morbidity and mortality (1, 2). In the united states, recent advances in the development of interferon sparing, direct - acting antiviral agents (daa) suggest that most treated patients can be cured, but given the limited access and high cost of daa on a global scale, prevention of infection by effective vaccination remains the best hope for virus eradication . However, in contrast to drug development, progress in vaccine development has been slow, in large part due to the extraordinary genetic diversity and rapid sequence evolution of the virus (37). Globally, hcv is represented by seven major genotypes (1 to 7) that exhibit nucleotide sequence diversity of as much as 30% (8). Within individual infected subjects it is estimated that up to 10 virions are produced daily in a typical infected individual (9), and based on an rna - dependent rna polymerase error rate of 2.5 10 per nucleotide per replication cycle (10), most of these are expected to be unique . In fact, every possible single point mutation, as well as every possible combination of two mutations, across the 10-kb viral genome are predicted to be generated every day (11). This explains the rapid appearance of resistance mutations to daas and the virus's capacity to evade host adaptive immune responses . Against this backdrop of nearly unfathomable viral diversity is substantial evidence indicating that hcv exhibits a relatively stringent population bottleneck at the moment of transmission from one individual to the next (1214). Such a transmission bottleneck is of clinical importance because at this point in viral natural history the infection is most vulnerable to treatment and prevention measures, including, potentially, vaccination (5, 1517). A virus bottleneck at transmission was initially inferred from clinical epidemiological studies that correlated risk of infection to the volume of blood exposure and the route of exposure (e.g., blood transfusion, injection drug use, needlestick injury, or mucosal inoculation, especially in hiv-1-positive men who have sex with men [1, 1823]). More refined estimates of the transmission bottleneck came from studies using a variety of increasingly sensitive and specific molecular techniques to characterize viral diversity, including oligonucleotide heteroduplex gel shift, vrna / cdna population sequencing, and molecular cloning, followed by sanger sequencing and next - generation deep sequencing (12, 13, 2432). These reports described the hcv transmission bottleneck in both qualitative and quantitative terms, but none allowed for a precise identification of transmitted or founder virus genomes . The most exacting quantitative estimates of the transmission bottleneck thus far came from studies based on single - genome sequencing (sgs), mathematical modeling, and phylogenetic inference (10, 14, 3335). This approach, which was adapted from hiv-1 infection studies (3639), theoretically allows for an unambiguous inference and quantitative estimation of transmitted / founder (t / f) viral genomes that are responsible for establishing productive clinical infection . The conceptual and mathematical models underlying this strategy assume a virus population emanating from each t / f genome that expands exponentially with no recombination, no selection pressure on variable sites, and a constant mutation rate across lineages (36, 40, 41). Under these conditions, the frequency distribution of the hamming distances (i.e., the pairwise distance between genetic strains) is given by a poisson distribution whose mean depends linearly on the number of generations since the founder strain (36, 40, 41). If the expansion is sufficiently rapid, small samples of sequences exhibit a star - like phylogeny whose coalescent corresponds to the t / f genome . For this situation, an analysis by forward simulation suffices, and a bayesian inference (e.g., beast [42, 43]), which is a backward simulation of genealogies, provides no advantages . Previously, we used the forward simulation model to analyze hcv sequences from 17 subjects in the very earliest stages of acute infection (weeks before hcv antibody seroconversion). Sequences from 13 subjects conformed well to model predictions, showing a star - like phylogeny and a poisson distribution of mutations within one or more distinct and well - separated lineages, each with low diversity (14). We interpreted these low - diversity sequence lineages to represent the progeny of discrete t / f genomes, thereby allowing for the identification and enumeration of the t / f genomes that resulted in productive clinical infection . In these 13 study subjects, the estimates of numbers of t / f genomes ranged from 1 to 13 per individual with a mean of 4 and a median of 3 (14). In the same study, however, viral sequences from four other acutely infected subjects (subjects 10003, 10016, 10020, and 106889) appeared to violate this forward simulation model by exhibiting multiple closely related lineages that failed to conform to a poisson distribution of mutations or star - like phylogeny . Some lineages differed from others by as few as one to three informative sites (shared mutations) out of the 5,000 bases sequenced . For these patients, our estimates for the numbers of t / f genomes were as high as 30 or more . We considered two possible explanations for these outlier results: either early hcv diversification in these subjects failed to follow a simple pattern of random diversification, creating multiple distinct but closely related lineages early on because of features unique to the hcv replication schema confounded by other unknown virus - host factors, or viral sequences in the donors to these four cases had undergone a population diversity bottleneck event prior to the transmission of multiple closely related variants, which then evolved as expected by the forward simulation model . Such a bottlenecking event in the donors could occur as a result of antiviral treatment, immunological selection or very recent acquisition of virus (i.e., the donors were themselves acutely infected). To explore these possibilities, we developed a different forward simulation model of early virus diversification that accounts for essential differences in replication dynamics between hcv and hiv-1 . Based on the patterns of shared mutations observed in these forward simulations, we developed a phylogenetically based clustering method that counts the number of putative t / f genomes (14; http://www.santafe.edu/~tanmoy/programs/hcv/). This approach thus incorporates the important features of hcv's unique cytosolic life cycle, where many replication complexes continuously produce virus from long - lived hepatocytes (10, 44, 45). Because of the sequential creation of as many as 40 replication complexes per cell (46), those at the same generation depth have widely varying numbers of descendants, unlike the situation in hiv-1 infection this leads to two predictions that differ substantially from the hiv-1 case: (i) sequence diversity of hcv saturates around the same time during primary infection that viral load stabilizes, instead of growing linearly in time as is the case for hiv-1, and (ii) there is an expectation of about three times as many stochastically shared mutations in hcv compared to hiv-1 infection when sampled in this stable phase, leading to more common deviations from a star - like phylogeny and poisson distribution of mutations (10, 14). When the second model was applied to sequences from subjects 10003, 10016, 10020, and 106889, it still failed to account fully for the atypical sequence diversification patterns and large numbers of potential t / f lineages observed in these subjects (14). We thus sought alternative explanations for the atypical early viral diversity, including the possibility of a pretransmission population bottleneck, followed by high - multiplicity virus transmission . In one case (subject 106889), we found evidence for such a bottleneck in the donor virus population due to prior treatment with a protease inhibitor (14). For subjects 10003, 10016, and 10020, however, there was no evidence of a drug - induced bottleneck, so we instead hypothesized that the donors for these subjects might themselves have been acutely infected or their viruses subjected to stringent immunological selection (32; unpublished data). In this scenario, the transmission of multiple viruses would be expected to yield a diversity pattern in the recipient comprised of very closely related viral lineages in addition to more distantly related lineages . In africa, it is estimated that acute - to - acute infection is responsible for as many as 25% of incident hiv-1 cases (4749). This can result in a pretransmission virus population bottleneck in the donor and acquisition of closely related viruses by the recipient (36, 38). Unfortunately, in our previous hcv studies (14), we had no documented cases of acute - to - acute virus transmission to evaluate this scenario . So, in the present study, we sought new examples of epidemiologically linked, sequence - confirmed, acute - to - acute hcv transmission . Here, we analyze early patterns of hcv sequence diversity in epidemiologically linked, viral sequence confirmed, donor - recipient, acute - to - acute transmission pairs, including human - to - human and human - to - chimpanzee infections . We tested two hypotheses: first, that hcv genomes responsible for transmission to a naive host can be identified unambiguously in paired donor - recipient plasma samples and that these sequences will be identical or nearly so, and second, that high - multiplicity infection of a subject with plasma from a donor who is acutely infected (and thus harbors sequences exhibiting a pretransmission population bottleneck) will recapitulate patterns of virus diversity observed in subjects 10003, 10016, and 10020 . In so doing, they validate the strategy of t / f genome identification by sgs, mathematical modeling, and phylogenetic inference for hcv, and they provide a plausible explanation for what had previously been a confusing set of hcv sequence data in a subset of acutely infected subjects . Our findings indicate that simple models of random virus diversification can generally explain early hcv evolution, although models that account for unique features of the hcv replication strategy offer a more conservative estimate of t / f genome numbers in samples that fail to conform to a star - like phylogeny and a poisson distribution of mutations . Our results illustrate how t / f analysis in hcv infection, much like t / f analysis in hiv-1 infection, represents a novel experimental strategy that can be used to molecularly anchor and uniquely inform studies of hcv natural history, immunopathogenesis, treatment, and prevention . The present study was conducted according to the principles expressed in the declaration of helsinki . It was approved by the institutional review boards of the university of pennsylvania and mt . Sinai medical center, new york, ny . Subjects provided written informed consent for the collection of blood samples and subsequent analyses . Chimpanzee specimens were collected and stored as part of a previously conducted study (50), and thus the present research is in full compliance with national institutes of health (nih) policies (not - od-12 - 025) concerning biomedical research involving chimpanzees . The subjects either were regular source plasma donors (zeptometrix, inc . ; seracare, inc .) Who were hcv and hiv-1 antibody negative and became hcv infected sometime in the course of their twice - weekly plasma donations, as evidenced by the development of hcv viremia on sequential viral rna testing, or they were hcv antibody - negative patients who became acutely infected with hcv and sought medical consultation . Chimpanzee inoculations with hcv - infected human plasma, subsequent specimen collections, and analysis of plasma virus load and liver specific transaminase levels were previously reported (50). Plasma samples were tested for hcv rna and antibodies by a battery of commercial tests . These included the roche molecular systems cobas amplicor hcv monitor (v2.0) assay, the abbott anti - hcv 3.0 assay, and the ortho anti - hcv 3.0 enzyme - linked immunosorbent assay . Additional vrna analyses using the transcription - mediated amplification (tma) method were performed as described previously (50). For each plasma sample, approximately 100,000 viral rna copies were extracted using the qiagen biorobot ez1 workstation with an ez1 virus minikit v2.0 (qiagen, valencia, ca). Reverse transcription of rna to single - stranded cdna was performed using mulv (superscript iii) reverse transcriptase (rt; invitrogen life technologies, carlsbad, ca) according to the manufacturer's instructions . Briefly, each cdna reaction included 1 rt buffer, 0.5 mm concentrations of each deoxynucleoside triphosphate, 5 mm dithiothreitol, 2 u of rnaseout (an rnase inhibitor)/l, 10 u of superscript iii reverse transcriptase/l, and 0.25 m antisense primer . The cdna primer used for amplifying each genome fragment corresponded to the first - round antisense primer listed in the single - genome amplification section below . The reverse transcription reaction was carried out at 50c for 60 min, followed by an increase in temperature to 55c for an additional 60 min . The reaction was then heat inactivated at 70c for 15 min and then treated with 0.1 u of rnase h/l at 37c for 20 min . The sequences from all of the subjects were generated using the sgs method previously described (14). Nearly full length, 5 half, 3 half, or partial ns2, ns3, and ns4a genomes were amplified for each subject by nested or seminested pcr, respectively . Cdna was serially diluted and distributed among wells of replicate 96-well plates (applied biosystems, foster city, ca) so as to identify a dilution where pcr - positive wells constituted less than 30% of the total number of reactions . At this dilution, this was confirmed in every positive well by direct sequencing of the amplicon and inspection of the sequence for mixed bases (double peaks), which would be evidence of priming from more than one original template or the introduction of pcr error in early cycles . Pcr amplification was carried out in the presence of 1 high - fidelity platinum pcr buffer, 2 mm mgso4, 0.2 mm concentrations of each deoxynucleoside triphosphate, 0.2 m concentrations of each primer, and 0.025 u of platinum taq high - fidelity polymerase/l in a 20-l reaction (invitrogen, carlsbad, ca). Nearly full length, 5 half or partial ns2, ns3, and ns4a genomes were amplified . The primers used were as follows: (i) subject 110069, first - round sense primer 1a.core.f1 (5-gcacgaatcctaaacctcaaagaaaaa-3; nucleotides [nt] 346 to 372, h77), first - round antisense primer 1ans5bend.r1a (5-ccggagtgtttatcccaaccttcat-3; nt 9374 to 9398, h77), second - round sense primer 1a.core.f2 (5-cgggtggcggtcagatcgttggtggagttta-3; nt 415 to 445, h77), and second - round antisense primer 110069.ns5b.r2a (5-gcggccgctattggagtgagtttgag-3; nt 9201 to 9226, h77); (ii) bgi and cci, first - round sense primer 1b.core.f1 (5-atgagcacgaatcctaaacctcaaaga-3; nt 342 to 368, h77), first - round antisense primer ns4a_r1 (5-gcactcttccatctcatcgaactc-3; nt 5451 to 5474, h77), second - round sense primer 1b.core.f2 (5-tcaaagaaaaaccaaacgtaacaccaaccg-3; nt 362 to 391, h77) and second - round antisense primer ns4a_r2 (5-aggtgctcgtgacgacctccagg-3; nt 5297 to 5319, h77); (iii) subjects 10081 and x355, first - round sense primer 10081ns2_f2 (5-acccgaccctgatatttgatatcacc-3; nt 2983 to 3008, h77), first - round antisense primer 1ans4b.r3 (5-tattgtatcccactgatgaagttccacat-3; nt 5634 to 5662, h77), second - round sense primer 10081ns2_f3 (5-aaagtaccctactttgtgcgcgt-3; nt 3063 to 3085, h77), and second - round antisense primer 1ans4b.r4 (5-agggccttctgcttgaactgctc-3; nt 5517 to 5539, h77); and (iv) subjects 10083 and x331, first - round sense primer 10083stf1 (5-gacatcactaagctgctgatag-3, first - round antisense primer 3av2r2 (5-ttacttccagatcagctgaca-3), second - round sense primer new10083stf2 (5-cccgttatatttaatacaggcta-3), and second - round antisense primer 3av2r2 (5-ttacttccagatcagctgaca-3). Pcr amplicons were directly sequenced by cycle - sequencing using bigdye terminator chemistry and protocols recommended by the manufacturer (applied biosystems). Sequencing reaction products were analyzed with an abi 3730xl genetic analyzer (applied biosystems). Individual sequence fragments for each amplicon were assembled and edited using the sequencher program 5.0 (gene codes; ann arbor, mi). Inspection of individual chromatograms allowed for the identification of amplicons derived from single versus multiple templates . The absence of mixed bases at each nucleotide position was taken as evidence of amplification from a single viral rna / cdna template . This quality control measure enabled us to exclude from the analysis amplicons that resulted from pcr - generated in vitro recombination events or taq polymerase errors and to obtain multiple individual sequences that proportionately represented those circulating hcv virions . All of the sequences alignments were initially made with clustal w and then hand - checked using geneious 7.1.9 to improve the alignments according to the codon translation . Eight hundred three quarter genomes, 103 5 half genomes and 114 near full - length genomes were generated by sgs from seven human and chimpanzee subjects for the diversity analysis . Among the 1020 amplicons generated, the sequences of 928 were unambiguous at every position . Nine near - full length genomes had mixed bases at 1 to 2 positions per sequence . We inferred that these mixed bases resulted from taq polymerase errors in the early pcr cycles because the mixed bases represented only a subset of the polymorphisms in any one sequence . These sequences were retained in the analysis and mixed bases interpreted as lineage consensus nucleotides . The other 83 amplicons contained double peaks, clearly resulting from amplification from more than one template, and these sequences were excluded from the analysis . Sequences were analyzed using phylogenetic tree analysis together with a sequence visualization tool, highlighter (www.hiv.lanl.gov), that allows tracing of common ancestry between sequences based on individual nucleotide polymorphisms . Phylogenetic trees were generated by maximum - likelihood methods using phyml (51). For subjects productively infected by more than one t / f virus, lineages containing 3 or more closely related sequences were included in the lineage diversity analyses . One was a forward simulation of early viral expansion (36, 40, 41), and the second, a phylogenetic - based clustering method that accounts for essential differences in replication dynamics and replication mechanisms hcv and hiv-1 (14; http://www.santafe.edu/~tanmoy/programs/hcv/). Lineage - specific sequences were analyzed by the poisson fitter program (www.hiv.lanl.gov), which computes the best fitting poisson distribution through maximum likelihood, performs a goodness of fit test, and tests for star - phylogeny . Power calculations to estimate the likelihood of detecting rare sequence variants based on sampling depth were performed using methods previously described (see fig . S9 and the associated description in reference 36). Acute hcv infection has historically been defined as the first 6 months of infection, which reflects the natural history and immunopathogenesis of disease where a minority of individuals spontaneously resolves the infection by this point, while most patients, if left untreated, exhibit persistent viremia (1, 18, 52). Whether or not an individual clears virus depends on host immunogenetics, innate and adaptive immune responses, and viral genotype, as well as other factors (1, 5355). Figure 1 illustrates viral dynamics and diversity of plasma vrna / cdna sequences amplified as nearly full length (8.8 kb) genomic amplicons in a typical subject (e.g., 110069) experiencing acute (primary) hcv infection . In this case, overall viral diversity during the acute infection period was dictated far more by the number of transmitted viruses and the genetic distances among them (0.15 to 0.47%) than by early sequence evolution, which can undergo positive or negative selection, as well as population diversity bottlenecking (13, 14). Very early in acute infection prior to antibody seroconversion, sequences appeared as four readily distinguishable, low diversity lineages (t / f lineages 1 to 4). Within each lineage, sequences differed by no more than 3 nucleotides (nt) out of nearly 9,000 nt (0.03%), and exhibited a star - like phylogeny and a poisson distribution of mutations (table 1). Furthermore, the diversity within each lineage was roughly the same, as would be expected if all of the lineages had been diverging at about the same rate and for the same time . The pattern within each lineage conformed to a simple model of random virus evolution and is typical of most acute hcv infections (14). We inferred from these data that the coalescent of each low - diversity lineage corresponded to a distinct t / f genome in total numbering four . This is a minimum estimate because deeper sequencing could potentially detect variants present at lower abundance . In this case, 48 sequences from days 2 and 17 following the first plasma vrna positive time point were initially analyzed . Power calculations (36) predict that at this level of sampling, there is greater than a 90% likelihood of detecting minor variants present at a frequency of 5% . However, in data not shown or reported elsewhere (33), we used other primer sets and plasma rna samples from early time points to generate 228 additional overlapping 5 and 3 half genomes from this subject, giving a total of 276 sequences evaluated for diversity and phylogeny . At this level of sampling, power calculations indicate a> 90% likelihood of detecting minor variant lineages present at a frequency of 1% . Still, these are minimum estimates for the number of t / f genomes, and we cannot exclude the possibility that a sequence such as 110069_2f27 (situated between variants 3 and 4 in panel a) represents a distinct t / f genome or is an example of homoplasy . Nor can we exclude the possibility that next - generation deep sequencing could detect additional t / f lineages present at exceedingly low frequencies, although this has generally not been seen in acute hiv-1 infection (56, 57). Hcv sequence diversity in acutely infected human subject 110069 . A maximum - likelihood (ml) mid - point rooted phylogenetic tree (a) and highlighter plots (c and d) of nearly full length plasma vrna sequences spanning the initial 6-month infection period are depicted . Plasma viral load kinetics (b) are also depicted, and the sequences are color coded to correspond to sampling time points determined from the first vrna - positive / antibody - negative time point (blue, day 2; red, day 17; green, day 142). Hcv antibody tests were negative through day 17 but were positive at the next sampled time point on day 146 . The tree and the highlighter plot reveal productive clinical infection by four t / f viruses (t / f lineages 1 to 4). Viral diversity is indicated by the scale bar and bootstrap values are shown . In the highlighter (c), tick marks are color coded to indicate nucleotide substitutions by a (green), t (red), c (blue), or g (orange) compared to the consensus sequence of the first time point (tp1). In panel d, tick marks denote synonymous (green) or nonsynonymous (red) substitutions . Statistical analysis of hamming distance frequency distribution and star - like phylogeny poisson - fitter (www.hiv.lanl.gov) computes the poisson distribution by maximum - likelihood and chi - square goodness - of - fit analyses and tests for star - like phylogeny . The sequences from subject 10081 exceeded the diversity limits of the poisson fitter and thus did not conform to a poisson distribution or a star - like phylogeny . Pt1, patient 1; pt2, patient 2 . A goodness - of - fit (gof) p value of> 0.05 indicates a nonsignificant divergence from a poisson distribution . Approximately 5 months into infection, the pattern of sequence diversity in subject 110069 plasma was much different than at earlier time points (fig . There was evidence of a stringent population genetic bottleneck between the second and third sampling time points (days 17 and 142 in fig . The closer similarity of the 5-month sequences to t / f lineage 4 sequences (0.1% mean diversity) compared to t / f lineages 1 (0.5% mean diversity), 2 (0.6% mean diversity), or 3 (0.3% mean diversity) suggested that the 5 month sequences had all evolved from the t / f 4 lineage, with the descendants of t / f lineages 1 to 3 lost in a population / diversity bottleneck . Later sequences emanating from the t / f 4 lineage contained many shared mutations violating both a star - like phylogeny and a poisson distribution (table 1). Figure 1 (panel d) suggests that the population bottleneck likely resulted from immune selection since the highly selected nonsynonymous mutations in e2 (see vertical red stripe in fig . 1d) map to a previously identified cytotoxic t lymphocyte (ctl) epitope (58) and are adjacent to a contact residue of the broadly neutralizing antibody ar3c (59). In addition, the three strongly selected nonsynonymous mutations in ns3 map to previously identified ctl epitopes (58, 60, 61). Unfortunately, peripheral blood mononuclear cells were not available from this subject for human leukocyte antigen analysis or phenotypic testing to confirm phenotypic t cell recognition and escape of these and other potential epitopes . The patterns of sequence evolution observed here for subject 110069 illustrate how high - multiplicity transmission of very early acute infection viruses or viruses arising following an immune - mediated population bottleneck could lead to sequence patterns in a recipient that resemble what was seen in the earlier study's acutely infected outlier subjects 10003, 10016, and 10020 (14). To further test the validity of hcv t / f genome inferences, we next studied epidemiologically linked human - to - human and human - to - chimpanzee donor - recipient hcv transmission pairs in an attempt to document transmission of actual virus genomes and to study their subsequent evolution . Figure 2 illustrates plasma vrna kinetics and 80 5 half genome sequences from two human subjects (patient 1 [bgi], donor; patient 2 [cci], recipient), who became acutely infected with hcv at about the same time . These two individuals were hiv-1-positive men who have sex with men, who were regular sexual partners, who denied injection drug use (idu), and who were hcv antibody negative at the time of first vrna detection . The clinical history indicated that patient 1 first became symptomatic with acute hcv infection (fatigue, jaundice, elevated liver transaminases) leading to hcv screening of himself and his partner patient 2 by viral rna assay . Both individuals were confirmed to be positive for hcv rna and negative for hcv antibody . Figure 2 shows that patient 1 and patient 2 each harbored single low - diversity hcv sequence lineages that exhibited a near star - like phylogeny and a poisson distribution of mutations (table 1), indicating that each had been productively infected by a single t / f virus . Surprisingly, when the viral sequences from these individuals were analyzed together, they were found to be identical or nearly so . Whether the sequences were analyzed separately or together, they coalesced phylogenetically to the same t / f virus genome (fig . 2). To be certain that a sample mix - up had not occurred, which could explain the identity or near identity of their sequences, five additional plasma samples taken from these subjects in subsequent weeks were analyzed . A total of 162 sequences from patients 1 and 2 spanning a 15-week period all coalesced to the same single t / f genome . This confirmed that donor and recipient had both been productively infected by viruses containing an identical 5 half - genome sequence . Given the clinical history, the most plausible explanation for this result is that patient 1 became acutely infected by a single t / f virus, and very early in his acute infection period, when most of his circulating virus still contained no mutations, he transmitted a single virus to patient 2 . A less likely scenario is that patient 1 and patient 2 each acquired their virus from a third individual . This interpretation was not consistent with the clinical history and would require that this third individual also be acutely infected so as to transmit identical viruses to two partners . Either way, acquisition of viruses containing identical genomes by patient 1 and patient 2 corroborates the t / f genome strategy and the conclusion that early virus evolution generally follows a simple model of random sequence diversification . Hcv plasma vrna kinetics and 5 half genome hcv sequences from an epidemiologically linked human hcv transmission pair . (a) viral load profiles in the presumed donor (patient 1 [bgi]) and recipient (patient 2 [cci]) are illustrated . The sampling time points are circled and color coded red (donor) and blue (recipient). (b) ml phylogenetic trees and highlighter plots show that each subject was productively infected by a single virus . (c) the combined tree and plot show that the t / f viral genomes infecting both subjects were identical in sequence . We next sought to evaluate the t / f strategy in situations of low and high multiplicity infection from a transmitting donor who was acutely infected . This was done to determine whether hcv sequence patterns similar to those found in subjects 10003, 10016, and 10020 could be recapitulated and explained on the basis of a pretransmission virus population bottleneck . Because we had no access to additional epidemiologically linked human - to - human transmission pairs, we studied hcv transmission in the closely related human - to - chimpanzee infection model . Biomedical research in chimpanzees is now restricted, so we analyzed stored plasma samples from a previous study by busch et al . (50) that was designed to evaluate the clinical infectivity of human plasma sampled from the earliest phases of infection before plasma hcv vrna could be detected by u.s . Food and drug administration (fda)-licensed diagnostic assays . In those experiments, closely spaced sequential plasma samples from individuals who subsequently became acutely infected by hcv were inoculated into virus - naive chimpanzees to determine whether they contained infectious hcv . This study plan provided an ideal experimental design and sets of well - pedigreed frozen plasma samples from linked donors and recipients to test the concept of t / f genome identification under conditions of low- and high - multiplicity infections . The human donors for these studies were hcv antibody - negative source plasma donors who had donated plasma once or twice weekly for many months until, unexpectedly, they were found to have become positive for plasma hcv rna . In both human subjects whom we studied, plasma hcv rna increased rapidly and plateaued at high levels, followed weeks later by hcv antibody seroconversion in patterns typical of acute (primary) hcv infection . Figure 3a depicts plasma vrna kinetics of the acutely infected human donor 10081 (50) and shows the time of sampling for our hcv sequence determinations . The first plasma sample positive for hcv rna by an fda - licensed assay (roche molecular systems cobas amplicor hcv monitor pcr assay, v2.0; detection limit of 600 iu / ml or 2,040 vrna copies / ml) is designated as day 0 so as to maintain consistency with the previous report (50). Samples for day 21 through day 4 all tested negative for hcv rna by this fda - licensed viral load assay . A more sensitive transcription - mediated amplification (tma) assay (gen - probe / hologic, inc . ; detection threshold of 12 vrna copies / ml) was then used to test replicate plasma samples . Samples from days 21, 18, 14, and 11 were determined to be negative for vrna by this ultrasensitive test, but a subset of the replicates from days 7 and 4 was positive . Samples (50 ml) of plasma from day 18 through day 7 were infused into a virus - naive chimpanzee but did not lead to productive hcv infection . However, intravenous infusion of 50 ml of subject 10081 plasma from day 4, which was estimated to contain 60 (95% confidence interval, 25 to 95) vrna copies based on the tma assay, led to productive infection of chimpanzee x355 . Plasma viremia in chimpanzee x355 was first detected 2 weeks after inoculation and remained variably positive through week 8 (fig . This pattern of early viremia, transient elevation of alanine aminotransferase (alt), followed by resolution of viremia is typical of chimpanzee hcv infection . Hcv viral load kinetics and sequences from a human - to - chimpanzee transmission pair . (b) fifty milliliters of 10081 plasma from the day 4 time point was infused intravenously into chimpanzee x355 at week 0 . Plasma vrna kinetics are indicated by filled black dots, with blue and green circles indicating time points subjected to sequence analysis . Alanine aminotransferase (alt) is a hepatic enzyme that when elevated above baseline indicates liver inflammation or injury . An ml phylogenetic tree (with sequences color coded red, black, blue, and green to correspond to time points indicated in panels a and b) and a highlighter plot show that the human subject 10081 was acutely infected by multiple viruses, one of which, h1 (top of panel c), was transmitted to chimpanzee x355 giving rise to the c1 t / f lineage . Note that the t / f genome corresponding to h1 is identical in sequence to the t / f genome corresponding to the c1 lineage . One hundred eighty - four human hcv sequences and 140 chimpanzee hcv sequences are shown (fig . This sequencing depth provided over 90% likelihood of detecting minor variants present at frequencies of 2% . Phylogenetic analysis suggested infection of the human subject 10081 by a large number of viruses . In 10081, widely divergent lineages were evident but so too were many additional lineages comprised of sequences that contained one, two, three, or more shared nucleotide polymorphisms . These multiple sets of very closely related sequences failed to conform to a simple model of random virus diversification and resembled those from subjects 10003, 10016, and 10020 (14). Using the revised models to account for differences in replication strategies of hcv and hiv-1 (14; http://www.santafe.edu/~tanmoy/programs/hcv/), the sequence pattern in subject 10081 (fig . 3c) could be explained by the acquisition of between 9 and 14 t / f viruses . These sequences exhibited a star - like phylogeny and a poisson distribution of random mutations (table 1). Importantly, the inferred t / f hcv genome in the acutely infected chimpanzee (t / f lineage c1) was found to be identical to one of the human t / f sequence lineages (h1). This result documents the transmission of an hcv genome unchanged from donor to recipient, again corroborating the t / f concept and the observation that early virus evolution generally follows a simple pattern of random sequence diversification . Figure 4 illustrates a second case of human - to - chimpanzee hcv transmission but at a much higher multiplicity of infection . 3, except that in this case, plasma samples from human donor 10083 from days 49, 7, and 5 (the sample immediately preceding frank viremia) failed to infect chimpanzee x331 despite containing 784 hcv vrna copies (50). To show that x331 was infectible by hcv, busch et al . Next inoculated this chimpanzee intravenously with 50 ml of the day 0 human plasma, estimated to contain 3.4 10 hcv vrna copies (50). Productive infection of chimpanzee x331 was detected 1 week later and persisted through week 16 (fig . We sequenced by sgs the human 10083 plasma samples from days 0 and 2 (fig . 4a) and chimpanzee x331 plasma samples from weeks 1, 8, and 10 (fig . 4d) were analyzed . As for human donor 10081, phylogenetic analysis of human donor 10083 sequences revealed multivariant hcv infection resulting in multiple sets of very closely and more distantly related sequence lineages . Using our previously described clustering model (14; http://www.santafe.edu/~tanmoy/programs/hcv/), we estimated that subject 10083 had been productively infected by at least 12 t / f lineages (h1 to h12). This is a minimum estimate because of sampling limitations and because lineages differing by as few as one or two shared mutations could represent distinct transmitted variants . The diversity among the 12 inferred t / f genomes ranged from as little as 0.1% between h1 and h2 to as much as 0.6% between h11 and h12 (fig . 4d), reflecting the sequence diversity in the individual from whom 10083 acquired hcv infection . The phylogeny of sequences from chimpanzee x331 was quite different from that observed in chimpanzee x335 where only a single t / f lineage was identified . Instead, in chimpanzee x331, we found evidence by the clustering model (14; http://www.santafe.edu/~tanmoy/programs/hcv/) of a minimum of 10 t / f lineages (c1 to c10), with an overall pattern of diversity closely resembling that of subject 10083 hcv sequences (compare fig . 4c and d). When 10083 and x331 sequences were combined, phylogenetic analysis confirmed their close similarity (fig . 4f). In order to analyze this combined sequence set for unequivocal evidence of transmission of sequences from donor to recipient, we selected all sequences that were identical between 10083 and x331 and analyzed them in a combined phylogenetic tree (fig . We identified 13 discrete t / f lineages in chimpanzee x331 that had matching identical sequences in the human donor 10083 . This was three more t / f genomes (c11, c12, and c13) than were inferred by the clustering model (fig . The 13 t / f genome lineages thus correspond to viral genomes that were transmitted unaltered from the human donor 10083 to chimpanzee x331 . This is likely an underestimate of the numbers of t / f viruses giving rise to productive infection in chimpanzee x331, given the limitations of sampling and the very high virus inoculum . Nonetheless, the unequivocal identification of 13 t / f genomes that were identical in donor and recipient again corroborates the t / f concept for hcv and recapitulates the patterns of sequence diversity that we observed previously in subjects 10003, 10016, and 10020 (14). Hcv viral load kinetics and sequences in a human - to - chimpanzee transmission pair . (b) fifty milliliters of 10083 plasma from the day zero time point was infused intravenously into chimpanzee x331 at week 0 . Plasma vrna kinetics are indicated by filled dots, with blue, purple, and green circles indicating time points subjected to sequence analysis . (c) an ml phylogenetic tree (with sequences color - coded red and black to correspond to time points indicated in panel a) and a highlighter plot show that the human subject 10083 was acutely infected by multiple viruses . The model described by bhattacharya and coworkers (14; http://www.santafe.edu/~tanmoy/programs/hcv/) suggests a minimum of 12 t / f genomes (h1-h12). Panel d depicts sequences (color coded blue, purple, and green to correspond to time points indicated in panel b) from chimpanzee x331, and the bhattacharya model suggests a minimum of 10 t / f viruses (c1 to c10). (e) human (red and black) and chimpanzee (blue, purple, and green) sequences are combined . (f) an ml tree of sequences found to be identical between the human donor and the chimpanzee recipient . This tree reveals 13 t / f genome lineages (c1 to c13) that were transmitted unaltered from humans to chimpanzees . These 13 transmitted genomes are enclosed in black ovals in panel d and include three lineages (c11, c12, and c13) that were identified empirically (f) but not by the bhattacharya clustering analysis (d). The findings of the present study provide an essential validation of the t / f strategy, originally developed to identify transmitted hiv-1 genomes (36, 38, 39, 62, 63), for hcv . The results distinguish modes and multiplicities of virus transmission (14, 35) and mathematical models of early virus diversification and evolution (10, 14, 36, 40, 41; http://www.santafe.edu/~tanmoy/programs/hcv/). 1) (14) and cases of human - to - human (fig . Transmission where hcv sequences in donors and recipients were found to be identical, thus proving that the inferred t / f viral genomes in these subjects corresponded to actual transmitted viruses . These findings further support the conclusion that hcv sequence evolution in the initial weeks following virus transmission generally conforms to a simple model of random virus evolution with sequences characterized by a within - lineage star - like phylogeny and a poisson distribution of mutations . This allows for precise estimates to be made of numbers of t / f genomes associated with different clinicoepidemiological circumstances or risks of hcv transmission such as needlestick injury, blood transfusion, injection drug use, mucosal exposure, and acute - to - acute infection outbreaks (1, 14, 1823, 28, 35, 50, 64), where the numbers of t / f viruses responsible for productive clinical infection may vary widely (14, 35). These observations are important for future studies aimed at molecularly characterizing viruses responsible for infection, coinfection, superinfection and reinfection (64), for analyzing the selective or sieving effects of neutralizing antibodies and virus specific t cells that emerge postinfection or postvaccination (6569), and for analyzing the transmission or emergence of drug - resistant mutants (14). But if identification of t / f genomes is straightforward in cases where sequences conform to a simple model of random virus diversification, how can the present results explain the atypical examples previously described where acute infection sequences show very closely related lineages that appear to violate neutral evolution (i.e., subjects 10003, 10016, and 10020 in reference 14) and subjects 10081 and 10083 in the present study? The current data show that in cases where a pretransmission virus population diversity bottleneck is present in a donor (e.g., subject 10083 in fig . 4c) and the multiplicity of infection is high, the resulting pattern of virus diversity in the recipient (chimpanzee x331 in fig . 4d) can mirror that in subjects 10003, 10016, and 10020 (14). A pretransmission bottleneck can result from acute infection of the donor, treatment of the donor with antiviral drugs, or, in some instances, potent immune selection by neutralizing antibodies and/or ctls . If instead, a low - multiplicity infection occurs from a similarly bottlenecked donor (e.g., subject 10081 in fig . 3c), sequence diversity in the recipient (chimpanzee x335) follows the predicted random pattern with star - like phylogeny and a poisson distribution of mutations (fig . These findings suggest that during acute infection, the majority of distinct sequence lineages reflect diversification from discrete, and in some cases, very closely related t / f genomes . A star - like topology is expected to persist, while the majority of the replication complexes present result from a rapid burst of diversification, probably up to 6 weeks or so postinfection when an acquired immune response is typically first detected . The 13 t / f genome lineages in chimpanzee x331 support this conclusion (fig . However, in the absence of sgs data from linked donors and recipients, the complex patterns of closely related, low diversity sequence lineages such as shown for 10081 (fig . 4d), cannot all be assumed to represent distinct t / f genomes, and it is impossible to determine with certainty which were transmitted and which evolved posttransmission as a consequence of early stochastic mutations that then persisted in the population . Such complex data sets can be evaluated more conservatively by a relaxed deterministic model of hcv diversification (14; http://www.santafe.edu/~tanmoy/programs/hcv/). In the case of chimpanzee x331, we could evaluate how closely the approximations of t / f lineages based on this mathematical model corresponded to t / f lineages documented empirically: 10 by the former, 13 by the latter (fig . An interesting question is why, given the complex replication strategy of hcv, early evolution of individual t / f genome lineages does not violate the predictions of a simple model of random diversification more frequently . The likely explanation is that in acute hcv infection, plasma virus titers, the numbers of infected hepatocytes, and the numbers of intracellular replication complexes all increase exponentially and proportionately until plateau viremia is reached . For a fast enough exponential expansion rate, at each generation the progeny of new replication complexes dominate the virus population and, as a result, the phylogenetic tree of a small sample of viruses shows few coalescent events at generations much after the common ancestor . In this situation, hcv diversification resembles that of a fast - growing hiv-1 viral population, and the long - lived nature of hcv replication complexes in untreated infection (10) does not modify the essentially random mutational patterns observed in early samples . During the plateau phase of viremia, the linear stamping press replication process from a relatively fixed number of infected hepatocytes (10) dominates and most replication complexes formed show evidence of this shared viral ancestry . Consistent with this picture, it was observed that the average sequence diversity in a sample grows almost linearly at very early times during the virus titer exponential growth phase and saturates later when the virus reaches the plateau phase (10, 14). Once adaptive immune responses, including neutralizing antibodies and cytotoxic t cells, emerge, the viral load decreases and nonrandom virus diversity becomes evident (fig . We presented examples of nearly full length (8.8 kb), 5 half (4.9 kb) and 5 quarter (2.4) genomic analyses . Nearly full length single - genome amplifications are not always possible because of priming and extension inefficiencies, time and cost considerations, and other factors . However, because hcv contains a nonsegmented rna genome, analyses of sequences of a reasonable length (2 to 5 kb) that contain sufficient numbers of informative sites (shared nucleotide polymorphisms) are sufficient to identify and distinguish distinct t / f genomes . In addition, for applications where the intent is to characterize particular gene products such as env (e1e2), a 2-kb 5 amplification product may be sufficient to identify t / f genomes, characterize population diversity bottlenecks, and identify mutational escape patterns . Where the intent is to map on a proteome - wide basis all t cell epitopes recognized or escaped, nearly full length amplification is required in order to maintain genetic linkage across all alleles, as we have described previously for hiv-1 (37, 70). There are additional caveats to the extrapolation of the findings reported here to other cases of acute hcv infection and to different risk groups . First, we studied relatively few subjects, and for most of these individuals, risk behaviors leading to hcv infection were unknown . Future studies could benefit from an analysis of larger numbers of human subjects with more clearly defined risk factors for hcv acquisition (e.g., needlestick, blood products, idu, sexual, and other). Second, our models of early virus replication and diversification of hcv (14), as for hiv-1 (36), cannot fully account for the possibility of extremely early mutations that occur during one or more of the initial replication cycles of the incoming virus (for hcv, plus - sense rna to minus - sense rna to plus - sense rna, etc . ). This can result in the early occurrence and persistence of shared mutations, deviations from a star - like phylogeny, and inference of a t / f genome one or a few virus generations subsequent to the actual transmission event . In practice, very early mutations resulting in shared polymorphisms are uncommon in acute hcv vrna sequence data sets (14, 33), and when they do occur, they do not confound the inference of founder genomes . The paucity of very early mutations persisting to later virus generations may be due in part to a lower in vivo error rate of the hcv polymerase than that estimated from in vitro analyses (10) and because random mutations are generally neutral or deleterious and subject to early purifying selection (71). Thus, while it will not always be the case that the consensus of a low - diversity sequence lineage that conforms to model predictions represents the transmitted genome, such a sequence is nonetheless a founder sequence likely to have arisen within one or few virus generations of the transmitted genome . The phenotypic properties of such founder viruses, including their antigenicity, is likely to be equivalent to that of the corresponding transmitted viruses because of the short time interval and small numbers of virus generations that separate the two . What are the implications of the present findings for future hcv research? Because of the exceedingly short half - life of hcv in human plasma (t1/2 45 min) (72), plasma virus titers and genetic composition closely reflect the spectrum of viruses replicating in hepatocytes, along with the composite effects of biological, immunological and antiviral drug pressures influencing virus replication and persistence . As a consequence, quantitative analyses of plasma virus titers and genetic composition provide an extremely sensitive and precise real - time indicator of biologically relevant, and oftentimes clinically relevant, virus - host interactions . The current findings show how sgs of hcv genes and nearly full length genomes can inform important areas of hcv research . For example, with the recent development and clinical introduction of new curative treatment strategies, elucidation of treatment failures due to antiviral drug resistance, inadequate dosing or compliance, or virus reinfection becomes paramount . Sgs, which maintains genetic linkage within genes (e.g., ns5b in patients receiving polymerase inhibitors) and across genomes (e.g., ns3-ns5b in patients receiving protease, ns5a, and polymerase inhibitors), can contribute uniquely to the elucidation of mechanisms of treatment failure, as described previously for hiv-1 infection (73). Similarly, distinguishing hcv coinfection from superinfection or reinfection can be important in explaining apparent cases of treatment failure . This can best be done by sgs, as suggested by irving and brown (28, 64) and by the findings reported here . In the area of vaccine development, sgs has been uniquely informative and represents a major tool for assessing virus responses to host adaptive immunity in natural hiv-1 infection and following vaccination . This has included proteome - wide analyses of t - cell responses (37, 70, 74), env gp160-wide analyses of neutralizing antibody responses (36, 75, 76), and sieve analyses of vaccine breakthrough cases (6568, 77). Current and future hcv vaccine trials (3, 4) are primed to adopt this methodology and the associated bioinformatic and statistical platforms to accelerate hcv vaccine development and testing . Comparable studies in other flavivirus or alphavirus systems may similarly benefit from a precise molecular identification of t / f genomes and their early molecular pathways of diversification.
Glucagon - like peptide 1 (glp-1) is an incretin secreted from the l cells of the gastrointestinal tract in response to nutrient ingestion . In healthy control subjects, its physiologic effects control glucose levels by stimulating glucose - dependent insulin secretion, inhibiting glucagon secretion, and delaying gastric emptying (1). Extensive studies of the mechanisms of glp-1 analogs in patients with type 2 diabetes have confirmed their physiologic actions (4,5). These drugs as well as dipeptidyl - peptidase iv (dppiv) inhibitors, such as sitagliptin, saxagliptin, linagliptin, alogliptin, and vildagliptin, which inhibit the degradation of glp-1, are routinely used for treatment of this form of diabetes (6,7). (8) have demonstrated a progressive rise in meal - stimulated glucagon response associated with declining endogenous insulin production . Glp-1 analogs have been shown to inhibit glucagon levels, and therefore insulin - deficient individuals with type 1 diabetes may show a beneficial response on the basis of reduced glucagon secretion . Moreover, animal studies have suggested that glp-1 therapy may promote the proliferation of -cells, enhance -cell recovery, and suppress -cell apoptosis (9,10), suggesting that there may be long - term primary benefit to its use . In patients with type 2 diabetes, glp-1 analogs have been shown to augment glucose - dependent insulin secretion (11,12), but the significance of this action is not clear inasmuch as detailed analyses of glp-1 receptor agonists in patients with residual insulin production are limited . Although older studies have highlighted the progression of type 1 diabetes to complete insulin deficiency, more recent studies have identified subjects with long - standing type 1 diabetes with residual insulin production (13,14). In these subjects, glp-1 analogs may improve glucose control and reduce the need for exogenous insulin (15), because there may be a significant functional component to the loss of insulin secretion, possibly due to -cell exhaustion from hyperglycemia (16). To assess whether combination therapies aimed at promoting -cell growth in addition to agents that decrease the autoimmune destruction of -cells, 20 subjects with long - standing type 1 diabetes were enrolled in a trial and randomized to exenatide with or without daclizumab (17). While c - peptide secretion increased with exenatide treatment, the difference failed to reach statistical significance . Other studies have suggested that residual insulin production is not a significant contributor to the effects of glp-1 receptor agonists . Infusion of glp-1 has been shown to reduce postprandial glycemic excursions in half in subjects with type 1 diabetes regardless of residual endogenous insulin production (18). However, this study was conducted with infusions of glp-1, and assessment was performed solely with orally administered glucose, which may not reflect physiologic stimuli . To determine the metabolic effects of glp-1 in patients with type 1 diabetes, we studied the acute effects of exenatide, a short - acting glp-1 analog, on glucose tolerance to a mixed - meal tolerance test (mmtt) and an intravenous glucose tolerance test (ivgtt). We analyzed insulin secretion rates (isrs), gastric emptying, and hormonal responses including glucagon, gastric inhibitory polypeptide (gip), and endogenous glp-1 release . We studied individuals with and without residual insulin production to determine the importance of insulin secretion on mediating the metabolic effects of the analog . We studied 17 patients with type 1 diabetes with (c - peptide positive) (n = 8) and without (c - peptide negative) (n = 9) residual insulin production who were recruited from the yale diabetes center . The presence of residual insulin production was identified post hoc after completion of the metabolic studies, and this information was used for the comparative data analysis . Patients were considered c - peptide positive if they had a c - peptide value of 0.017 nmol / l at any time point during the mmtt . To enhance our detection of individuals with residual insulin production, we recruited individuals between the ages of 18 and 56 years, with a diabetes duration of at least 2 years, hba1c level of <9%, and insulin usage of <0.9 units / kg / day . The study protocol was approved by yale university institutional review board . Written informed consent was obtained from all patients ., subjects received basal insulin (either as basal insulin through an insulin pump or as long - acting insulin); however, no short - acting insulin was administered for either the mmtt or ivgtt . The tests were performed in a randomized manner under two study conditions, with and without pretreatment with exenatide, 5 g s.c . A standard 4-h mmtt was extended to 5 h because previous studies had shown delayed gastric emptying with glp-1 use . For the mmtts, patients drank a liquid meal (boost high protein, 6 ml / kg), and blood samples were collected at 13 time points (10, 0, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 min) for the measurement of glucose, c - peptide, and glucagon, and, on a subset of subjects (n = 8), glp-1 and gip . Gastric emptying was evaluated by measuring the plasma levels of acetaminophen after a dose of 20 mg / kg to a maximum of 1,300 mg at the start of the meal . Ivgtts were performed by infusing a 20% dextrose solution at a dose of 0.5 g / kg up to a maximum of 35 g. blood samples for the measurement of glucose and c - peptide were collected at 10, 4, 1, 3, 5, 7, and 10 min . Glp-1 (n = 6) and gip (n = 4) samples were collected at the start and the end of the test . Plasma c - peptide levels were measured at northwest lipid metabolism and diabetes research laboratories (seattle, wa) using the tosoh aia 1800 assay . Nmol / l with intra - assay and interassay coefficients of variation (cvs) of 1.71 and 4.68%, respectively . The lower limit of detection was 19 pg / ml, with intra - assay and interassay cvs of 6.58 and 6.64% . Total glp-1 and gip levels were collected in tubes containing edta and dppiv inhibitor, and measured by elisa (alpco diagnostics and millipore). The interassay cvs for low and high glp-1 level controls are 8.8 and 7.0%, respectively, and for the low and high gip level controls were 9.2 and 8.1%, respectively . Acetaminophen was measured by roche modular p analyzer using syva emit reagents (siemens healthcare diagnostics ltd, newark, de). Plasma glucose levels were measured at the bedside using a biochemistry analyzer (model 2700 select; ysi and xylem). To determine isrs, the c - peptide levels obtained during mmtts and ivgtts were deconvoluted using a two - compartment model for hormone clearance with the chronobiological series analyzer (csa) software (19,20). Standard kinetic parameters for c - peptide were used based on the findings of van cauter et al . (21), who estimated rate constants based on extrapolations from c - peptide decay curves of 200 subjects . Parameters used for isr calculation accounted for the patient s age, sex, height, weight, and c - peptide values (in nanomoles per liter). The baseline insulin dose was calculated as the average number of units used per day for 3 days prior to the first visit . Subjects without detectable fasting c - peptide levels were classified as not having residual insulin production, and we did not observe detectable levels after the mmtt or ivgtt . Total area under the curve (auc) was calculated for isr, and glucose, glucagon, glp-1, gip, and acetaminophen levels using the trapezoidal rule . The isr / glucose ratio was calculated using the isr calculated from the time interval initiated with the glucose level . Statistical analyses were performed with graphpad (san diego, ca) prism version 5.04 . For comparison between the two groups, a wilcoxon matched - pairs signed rank test was used . Differences between groups resulting in two - tailed p values <0.05 were considered statistically significant . We studied 17 patients with type 1 diabetes with (c - peptide positive) (n = 8) and without (c - peptide negative) (n = 9) residual insulin production who were recruited from the yale diabetes center . The presence of residual insulin production was identified post hoc after completion of the metabolic studies, and this information was used for the comparative data analysis . Patients were considered c - peptide positive if they had a c - peptide value of 0.017 nmol / l at any time point during the mmtt . To enhance our detection of individuals with residual insulin production, we recruited individuals between the ages of 18 and 56 years, with a diabetes duration of at least 2 years, hba1c level of <9%, and insulin usage of <0.9 units / kg / day . The study protocol was approved by yale university institutional review board . Written informed consent was obtained from all patients . Subjects underwent two mmtts and two ivgtts . During all studies, subjects received basal insulin (either as basal insulin through an insulin pump or as long - acting insulin); however, no short - acting insulin was administered for either the mmtt or ivgtt . The tests were performed in a randomized manner under two study conditions, with and without pretreatment with exenatide, 5 g s.c . A standard 4-h mmtt was extended to 5 h because previous studies had shown delayed gastric emptying with glp-1 use . For the mmtts, patients drank a liquid meal (boost high protein, 6 ml / kg), and blood samples were collected at 13 time points (10, 0, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 min) for the measurement of glucose, c - peptide, and glucagon, and, on a subset of subjects (n = 8), glp-1 and gip . Gastric emptying was evaluated by measuring the plasma levels of acetaminophen after a dose of 20 mg / kg to a maximum of 1,300 mg at the start of the meal . Ivgtts were performed by infusing a 20% dextrose solution at a dose of 0.5 g / kg up to a maximum of 35 g. blood samples for the measurement of glucose and c - peptide were collected at 10, 4, 1, 3, 5, 7, and 10 min . Glp-1 (n = 6) and gip (n = 4) samples were collected at the start and the end of the test . The iv glucose bolus was given at 3 min over 3 min . Plasma c - peptide levels were measured at northwest lipid metabolism and diabetes research laboratories (seattle, wa) using the tosoh aia 1800 assay . Nmol / l with intra - assay and interassay coefficients of variation (cvs) of 1.71 and 4.68%, respectively . Glucagon levels were measured by radioimmunoassay (millipore, st . Charles, mo). The lower limit of detection was 19 pg / ml, with intra - assay and interassay cvs of 6.58 and 6.64% . Total glp-1 and gip levels were collected in tubes containing edta and dppiv inhibitor, and measured by elisa (alpco diagnostics and millipore). The interassay cvs for low and high glp-1 level controls are 8.8 and 7.0%, respectively, and for the low and high gip level controls were 9.2 and 8.1%, respectively . Acetaminophen was measured by roche modular p analyzer using syva emit reagents (siemens healthcare diagnostics ltd, newark, de). Plasma glucose levels were measured at the bedside using a biochemistry analyzer (model 2700 select; ysi and xylem). To determine isrs, the c - peptide levels obtained during mmtts and ivgtts were deconvoluted using a two - compartment model for hormone clearance with the chronobiological series analyzer (csa) software (19,20). Standard kinetic parameters for c - peptide were used based on the findings of van cauter et al . (21), who estimated rate constants based on extrapolations from c - peptide decay curves of 200 subjects . Parameters used for isr calculation accounted for the patient s age, sex, height, weight, and c - peptide values (in nanomoles per liter). The baseline insulin dose was calculated as the average number of units used per day for 3 days prior to the first visit . Subjects without detectable fasting c - peptide levels were classified as not having residual insulin production, and we did not observe detectable levels after the mmtt or ivgtt . Total area under the curve (auc) was calculated for isr, and glucose, glucagon, glp-1, gip, and acetaminophen levels using the trapezoidal rule . The isr / glucose ratio was calculated using the isr calculated from the time interval initiated with the glucose level . Statistical analyses were performed with graphpad (san diego, ca) prism version 5.04 . Data are presented as means se, unless indicated otherwise . For comparison between the two groups, differences between groups resulting in two - tailed p values <0.05 were considered statistically significant . We studied 17 subjects with type 1 diabetes with (n = 8) and without (n = 9) endogenous c - peptide secretion . Although all of the subjects had type 1 diabetes for at least 2 years, those with residual insulin production tended to have a shorter duration of diabetes (p = 0.11). The insulin production that was detectable in c - peptide positive subjects was responsive to the metabolic stimuli . Isrs increased approximately fourfold in those with residual insulin production during the mmtt without exenatide pretreatment, but only 1.3-fold in response to iv glucose (figs . 1 and 2c and d). Metabolic effects of exenatide during mmtt: the kinetics of isrs and glucose, acetaminophen, and glucagon levels are shown in a representative patient with (a d) or without (e h) detectable c - peptide levels . +, with;, without . Changes in hormonal responses during mmtt and ivgtt with exenatide.a: auc for glucose (in milligrams per deciliter) during an mmtt without and with administration of exenatide, 5 g s.c . B: auc for glucose (in milligrams per deciliter) during ivgtt without and with exenatide (p = 0.8501). C: auc for isr (in nanomoles per liter) during mmtt without and with subcutaneous injection of exenatide (p = 1.0). E: auc isr / auc glucose during mmtt with and without exenatide (p = 0.0078). F: isr auc / glucose auc at the time of ivgtt with and without pretreatment with exenatide (p = 1.0). G: auc for glucagon (in picograms per milliliter) during mmtt with and without administration of exenatide (p = 0.0015) in all subjects . H: auc of the glucagon / glucose ratio in all patients during mmtt, which shows a significant increase with exenatide (p = 0.006). Open circles () indicate c - peptide negative patients, and closed circles () indicate c - peptide positive patients . +, with;, without . The glucose, isr, glucagon, and acetaminophen responses of representative subjects with and without endogenous c - peptide secretion are shown in fig . When all subjects with and without residual insulin production were considered together, the glucose excursions after the liquid meal were reduced by 33% with exenatide pretreatment (without residual insulin production 79,626 3,869 mg / dl; with residual insulin production 4,665 3,427 mg / dl; p <0.001) (fig . We did not find a difference in the exenatide effects on glucose excursions in subjects with versus those without residual insulin production . In contrast, we did not detect an effect of exenatide on glucose responses to ivgtt (without residual insulin production 3,661 133.2 mg / dl; with residual insulin production 3,686 158.2 mg / dl; p = 0.8501) (fig . Exenatide is known to delay gastric emptying . To assess this effect in patients with type 1 diabetes, we measured the absorption of acetaminophen during the mmtt performed with or without exenatide pretreatment . Gastric emptying was delayed, and the total acetaminophen absorption was reduced from 2,058 196 to 686 138 g / ml (p = 0.0017). The effects on gastric emptying were also similar in subjects with and without residual insulin production . We analyzed the isr in the eight subjects with detectable levels of c - peptide . Of these, five subjects showed a reduction in the absolute isr auc when exenatide was given, and the remainder showed an increase . Thus, overall the absolute levels of isr were not changed by the exenatide (fig ., we did not see an effect of exenatide treatment on the insulin secretory response to ivgtt (fig . Interestingly, when we evaluated the relationship of isr to the glucose levels (auc isr / auc glucose) in c - peptide positive patients, we found that the levels were significantly higher in the exenatide - treated group (without treatment 0.1134 0.0388; with treatment 0.2318 0.08855; p = 0.0078) (fig . We did not see an effect of exenatide treatment on the insulin secretory response to ivgtt (without treatment 209.5 102.1 nmol / l; with treatment 297 217.1 nmol / l; p = 0.875) (fig . 2d) or an effect on the relationship between isr and glucose in c - peptide positive patients during the ivgtts (without treatment 0.0640 0.03675; with treatment 0.07925 0.05906; p = 1.0) (fig . Glucagon levels were significantly suppressed in the presence of exenatide (without treatment 15,909 945.8 pg / ml; with treatment 12,124 1,182 pg / ml; p = 0.0015) (fig . However, the glucagon / glucose ratio was significantly increased after exenatide treatment (without treatment 0.2116 0.01925; with treatment 0.2875 0.0446; p = 0.006), suggesting that the decrease in glucose excursion involved factors in addition to the effects on glucagon itself (fig . Using an assay that was specific for endogenous glp-1 and did not cross - react with the exenatide that was administered, we found that there were reduced glp-1 levels in six of the eight subjects, while the levels increased in two of eight subjects when they received exenatide, although the differences in the hormone levels did not reach statistical significance (without exenatide 662.6 270 pmol / l; with exenatide 444.2 302.1 pmol / l; p = 0.055) (fig . 3a). We did not find a significant change in the levels of gip when exenatide was administered (without exenatide 1,000 177.8 pg / ml; with exenatide 944.5 304.2 pg / ml; p = 0.875) (fig . 25.28 12.48 pmol / l vs. with exenatide 46.76 32.83 pmol / l) in six subjects (p = 0.625). D: auc for gip (in picograms per milliliter) during ivgtt (p = 0.875). We studied 17 subjects with type 1 diabetes with (n = 8) and without (n = 9) endogenous c - peptide secretion . Although all of the subjects had type 1 diabetes for at least 2 years, those with residual insulin production tended to have a shorter duration of diabetes (p = 0.11). The insulin production that was detectable in c - peptide positive subjects was responsive to the metabolic stimuli . Isrs increased approximately fourfold in those with residual insulin production during the mmtt without exenatide pretreatment, but only 1.3-fold in response to iv glucose (figs . 1 and 2c and d). Metabolic effects of exenatide during mmtt: the kinetics of isrs and glucose, acetaminophen, and glucagon levels are shown in a representative patient with (a d) or without (e h) detectable c - peptide levels . +, with;, without . Changes in hormonal responses during mmtt and ivgtt with exenatide.a: auc for glucose (in milligrams per deciliter) during an mmtt without and with administration of exenatide, 5 g s.c . B: auc for glucose (in milligrams per deciliter) during ivgtt without and with exenatide (p = 0.8501). C: auc for isr (in nanomoles per liter) during mmtt without and with subcutaneous injection of exenatide (p = 1.0). E: auc isr / auc glucose during mmtt with and without exenatide (p = 0.0078). F: isr auc / glucose auc at the time of ivgtt with and without pretreatment with exenatide (p = 1.0). G: auc for glucagon (in picograms per milliliter) during mmtt with and without administration of exenatide (p = 0.0015) in all subjects . H: auc of the glucagon / glucose ratio in all patients during mmtt, which shows a significant increase with exenatide (p = 0.006). Open circles () indicate c - peptide negative patients, and closed circles () indicate c - peptide positive patients . The glucose, isr, glucagon, and acetaminophen responses of representative subjects with and without endogenous c - peptide secretion are shown in fig . 1 . When all subjects with and without residual insulin production were considered together, the glucose excursions after the liquid meal were reduced by 33% with exenatide pretreatment (without residual insulin production 79,626 3,869 mg / dl; with residual insulin production 4,665 3,427 mg / dl; p <0.001) (fig . We did not find a difference in the exenatide effects on glucose excursions in subjects with versus those without residual insulin production . In contrast, we did not detect an effect of exenatide on glucose responses to ivgtt (without residual insulin production 3,661 133.2 mg / dl; with residual insulin production 3,686 158.2 mg / dl; p = 0.8501) (fig . Exenatide is known to delay gastric emptying . To assess this effect in patients with type 1 diabetes, we measured the absorption of acetaminophen during the mmtt performed with or without exenatide pretreatment . Gastric emptying was delayed, and the total acetaminophen absorption was reduced from 2,058 196 to 686 138 g / ml (p = 0.0017). The effects on gastric emptying were also similar in subjects with and without residual insulin production . We analyzed the isr in the eight subjects with detectable levels of c - peptide . Of these, five subjects showed a reduction in the absolute isr auc when exenatide was given, and the remainder showed an increase . Thus, overall the absolute levels of isr were not changed by the exenatide (fig ., we did not see an effect of exenatide treatment on the insulin secretory response to ivgtt (fig . Interestingly, when we evaluated the relationship of isr to the glucose levels (auc isr / auc glucose) in c - peptide positive patients, we found that the levels were significantly higher in the exenatide - treated group (without treatment 0.1134 0.0388; with treatment 0.2318 0.08855; p = 0.0078) (fig . We did not see an effect of exenatide treatment on the insulin secretory response to ivgtt (without treatment 209.5 102.1 nmol / l; with treatment 297 217.1 nmol / l; p = 0.875) (fig . 2d) or an effect on the relationship between isr and glucose in c - peptide positive patients during the ivgtts (without treatment 0.0640 0.03675; with treatment 0.07925 0.05906; p = 1.0) (fig . Glucagon levels were significantly suppressed in the presence of exenatide (without treatment 15,909 945.8 pg / ml; with treatment 12,124 1,182 pg / ml; p = 0.0015) (fig . However, the glucagon / glucose ratio was significantly increased after exenatide treatment (without treatment 0.2116 0.01925; with treatment 0.2875 0.0446; p = 0.006), suggesting that the decrease in glucose excursion involved factors in addition to the effects on glucagon itself (fig . Using an assay that was specific for endogenous glp-1 and did not cross - react with the exenatide that was administered, we found that there were reduced glp-1 levels in six of the eight subjects, while the levels increased in two of eight subjects when they received exenatide, although the differences in the hormone levels did not reach statistical significance (without exenatide 662.6 270 pmol / l; with exenatide 444.2 302.1 pmol / l; p = 0.055) (fig . 3a). We did not find a significant change in the levels of gip when exenatide was administered (without exenatide 1,000 177.8 pg / ml; with exenatide 944.5 304.2 pg / ml; p = 0.875) (fig . / l vs. with exenatide 46.76 32.83 pmol / l) in six subjects (p = 0.625). D: auc for gip (in picograms per milliliter) during ivgtt (p = 0.875). We studied the acute effects of exenatide, a short - acting glp-1 receptor agonist, in individuals with type 1 diabetes with and without residual insulin production . The primary objective of our study was to determine whether exenatide would affect the acute metabolic responses to an mmtt or an ivgtt in subjects with established type 1diabetes, and to determine the significance of residual insulin production on those responses . We calculated the isr rather than using raw c - peptide values, which gives a more accurate assessment of -cell function, inasmuch as the c - peptide levels may not reflect the true levels of insulin secretion because of the relatively long half - life of c - peptide . Moreover, we were able to study the effects of glp-1 analogs on endogenous glp-1 and gip in a subset of subjects . We found that administration of exenatide reduced glucose excursion and absolute glucagon secretion during an mmtt and delayed gastric emptying, similar to previous reports (2225). The total absorption of acetaminophen was reduced, and gastric emptying was delayed with exenatide administration . On average, we did not find an absolute increase in insulin secretion in subjects who were able to secrete insulin, but the relative amount of insulin secreted for the glucose was increased . Because we did not find an increase in the absolute amount of insulin that was secreted, the proportional increase in insulin for the glucose most likely is a reflection of the reduced glucose levels in subjects who were already maximally secreting insulin . The changes that we found in the glucagon / glucose ratio are consistent with the major effect on reducing glucose levels . Therefore, our studies indicate that the metabolic effects of exenatide involve two mechanisms, including its effects on the absorption of nutrients and glucagon inhibition, but based on our study design we cannot exclude an effect on augmenting insulin production as well . The metabolic effects require oral absorption of nutrients because we found no glycemic or hormonal effects of exenatide on the responses to intravenous glucose . In addition, the route of administration may be important because dppiv inhibitors have been shown to regulate glycemia by local inhibition of intestinal dppiv activity, activation of incretin receptors, and activation of the gut - to - pancreas neural axis (26). Endogenous glp-1 levels were decreased with exenatide administration in six of eight patients, which may have been due to the effects of the drug on gastric emptying or a feedback inhibition of glp-1 secretion (27). (15) studied the effects of exenatide prior to an mmtt in eight subjects with and eight subjects without residual insulin production . In these subjects, one - half of the usual dose of fast - acting insulin was administered together with exenatide . They reported that the incretin responses were similar in patients compared with healthy control subjects and found that the responses were also similar between those with and without residual insulin production . (28) studied hormonal responses in insulin - deficient patients with type 1 diabetes and found reduced insulin requirements and glucagon responses when glp-1 was infused . In addition to a similar reduction in glucagon release in response to a mixed meal that was seen by these previous authors, our data suggest a reduced level of glp-1 in most patients, but the number of subjects that we studied was small . Despite the profound effect of the glp-1 receptor agonist on glycemic excursion in our short - term studies, the effects of therapy with glp-1 receptor agonist on the clinical management of patients with type 1 diabetes have been relatively modest (29). The most significant metabolic consequence of exenatide administration appears to involve the delay in absorption of nutrients and the reduced rise in glucose as a consequence, but testing for additional benefits to individuals with residual insulin production may require further experience with larger numbers of subjects and longer use of an agonist . In addition to consideration of glp-1 receptor agonists in the chronic metabolic management, these agents might be considered in combination with other agents, including immune modulators in the new - onset period (30). In summary, in patients with long - standing type 1 diabetes, with and without residual insulin production, we observed a marked reduction in glycemic excursion during an mmtt with exenatide pretreatment, but no changes were observed in glucose excursion in response to an intravenous glucose challenge with exenatide pretreatment . In patients with residual insulin production, the insulin secretion was preserved even with reduced glucose levels . The value of glp-1 receptor agonists in long - term management of type 1 diabetes, particularly those with residual insulin production, will require long - term studies with larger numbers of subjects.
Peptidomimetics have found wide application as bioavailable, biostable, and potent mimetics of naturally occurring biologically active peptides . L - arginine has guanidinium group, which is positively charged at neutral ph and is involved in many important physiological and pathophysiological processes . It has a very ionizable amino acid, and it is found most frequently buried in the protein interior [25]. Arginine residues are essential in a variety of biological processes, such as the regulation of conformation or redox potentials [6, 7]; viral capsid assembly; electrostatic steering; voltage sensing across lipid bilayers [1012]; h+ transport [6, 1315] and peptide translocation across bilayers [16, 17]. They also play a critical role at protein - protein interfaces [5, 16], in enzymatic active sites [3, 5, 18], and in variety of transport channels [19, 20]. More recent findings show that arginine - specific methylation of histones may cooperate with other types of posttransitional histone modification to regulate chromatin structure and gene transcription . Proteins that methylate histones on arginine residues can collaborate with other coactivators such as nuclear receptors . They constitute nature's toolkit for making and breaking down the molecules required by cells in the course of growth, repair, maintenance, and death . Enzymes are capable of carrying out complex transformations in aqueous solution, at biological temperatures and ph, and in a stereospecific and regiospecific manner, a feat seldom achieved by the best of organic chemists [3, 22]. Crystallographic and nmr studies of enzymes have shed light on the relationship between an enzyme's three - dimensional (3d) structure and the chemical reaction it performs . There are many complications, however, in assigning the functions of catalytic residues, due to the multistep nature of a chemical reaction . One residue can play more than one role and can be involved in different steps of the reaction . Arginine (arg) is one of the most important residues in catalytic centers of many enzymes . Arg is in the 3rd place of the enzymatic frequency distribution and constitutes 11% of catalytic residues . Arg occurs more frequently than other basic residues (e.g., lysine) because it has three nitrogen containing groups in the side chain, all of which can perform electrostatic interactions . The side chain of arg can participate therefore in many electrostatic interactions, and it can be positioned more accurately to facilitate catalysis . The arg side chain has also a favorable geometry to stabilize a pair of oxygen atoms on a phosphate group (figure 1), a common biological moiety . Arg in a catalytic center can be involved in various kinds of interactions, for example, electrostatic, hydrogen bond formation, transition state stabilization, activation of water, and the activation of substrates . If the arg residue in the active site of the enzymes is replaced by an arg mimetic, this will cause the loss of enzymatic action, thus disturbing many metabolic pathways . Adenylate kinase (adk) was chosen as an example in order to explore how arg analogues will affect enzymatic action . This enzyme catalyzes the reversible reaction (1)2 adp atp+amp and may process metabolic signals associated with atp use [2328]. In this case, adk has been implicated in the regulation of the metabolically sensitive ion channels and transporters [2932]. In addition, disruption of the adk gene impedes the export of atp from the mitochondria . What will happen if a gene is working but some mutation of adk appears, such as arginine analogues (cav, ncav, ncan, nsarg, and sarg) being incorporated instead of arg in the active site of the enzyme? In order to study this issue, docking of analogues with arginyl - trna synthetase was our choice for the first step . This should show if analogues could be recognized by the enzyme responsible for transportation of arginine residues . Aminoacyl - trna constitutes a family of rna - binding proteins, that is, responsible for the correct translation of the genetic code by covalently linking the appropriate amino acid to the 3 end of the correct trna . In most organisms, there are 20 distinct aminoacyl - trnas, with each one of them being responsible for aminoacylating its cognate trna with unique amino acid in a two - step catalytic reaction . This intermediate interacts with appropriate trna molecule, and amino acid is transferred to 3 end of the trna . The protein sequence for human arginyl - trnasynthetase (hargs) was obtained from the uniprot database (accession number q5t160). Oxy- and sulfoarginine analogues: l - canavanine (cav), l - norcanavanine (ncav), l - norcanaline (ncan), l - norsulfoarginine (nsarg) and l - sulfoarginine (sarg), were previously synthesized and biologically tested [3639] (figure 2). In order to perform computational studies, the following software was used in the present work: compound preparation and homology modeling were done with molecular operating environment (moe); docking studies were performed by using genetic optimization for ligand docking (gold 5.1), run on the scientific linux 5.5 operating system; for generation of figures and the exploration of interactions after docking, the molegro molecular viewer (mmv) [43, 44] was applied . The best hit was proved with the crystallographic structure of arginyl - trna synthetase (args) of saccharomyces cerevisiae (protein data bank -pdb i d: 1f7u) with 40% identity . Five oxy- and sulfoanalogues of arginine with already known in vivo and in vitro biological effects were selected for docking studies . It uses a genetic algorithm and considers full ligand conformational flexibility and partial protein flexibility . The active site of hargs was defined as residues within 10 radius of glu130, which is responsible for guanidinium group recognition . One arginine residue seems to be very important at the active site of the enzyme arg175 is very close to the active site, and it could play a role in catalytic ability of the enzyme . Docking studies were performed with all mutated enzymes and bis(adenosine)-5-tetraphosphate in order to find out how mutations affect enzymatic action . The structure of adenylate kinase was obtained from rcsb (pdb i d: 2c9y). Homology modeling of hargs with a single template was performed by moe, and a standard molecular mechanics forcefield - amber99 was used . Phi - psi plot or ramachandran plot for the chosen model is presented on figure 3 . Disallowed regions are less than 1%, according to the ramachandran plot, so it could be used in further studies . It is known that atp binds to the loop close to the binding site of arginine . When there is no atp molecule attached to hargs, arginine binds as shown in figure 4(b). All the h - bonding capability of the substrate is used by the protein for the specific recognition . The -amino group of arginine forms h - bonds with main chain carbonyls of ser133 and phe134, while -carboxylate interacts with the amide nitrogen of asn135 and phenyl oxygen of tyr322 . Residues ser133 and asn135 are very important for correct recognition of -amino and -carboxyl groups . The guanidinium group forms two salt bridges with two carboxylate residues: glu130 and asp326 . If atp binds, some conformational changes occur, and -carboxyl group of arginine interacts with arg325, and this facilitates forming the arginyl - amp (figure 4(c)). Dockings with oxy- and sulfoanalogues of arginine were performed in order to check if they can act as substrates for this enzyme . The distance between its -carboxyl group and arg326 is too long to interact with it and thus to react successfully with atp (figure 5(a) and table 2). Nsarg has a sulfoguanidinium group, and though it is less polar than arginine's guanidinium group, interaction with glu130 is still present . In the case of this compound the carboxyl group interacts with other residues from the active site, and the distance between this group and arg325 is too long (figure 5(b)). In the case of sarg, the possibility for interaction between sarg and atp is higher than nsarg as its -carboxyl group is closer to arg325 (figure 5(b)). The enzyme can recognize ncav and cav as substrates, but only cav could interact with atp to form cav - amp . The distance between -carboxyl group and arg325 is too long to have a reaction with atp (figure 5(a)). From this series of arginine analogues, we could mark three of them that could play the role of substrates for hargs . These compounds could be incorporated in different peptides and protein molecules, and in the present work, we are analyzing the effects of this interaction on adenylate kinase . With the help of moe, we made mutations at two points subsequently in the active site of adk - arg138 and arg175, with cav, ncav, and sarg . After energy minimizations of 6 new structures, docking with bis(adenosine)-5-tetraphosphate was performed with gold . Bis(adenosine)-5-tetraphosphate could mimic the interactions in the binding sites of adk as it occupies both sites of the enzyme . For the recognition of purine moiety of the substrate, very important residue it interacts with all phosphate oxygen atoms of adp molecule by forming hydrogen bonds with backbone amide groups (figure 6). All three analogues of arginine have the guanidinium group as a structural element . In all three cases, it is connected via more electronegative atoms (oxygen and sulfur) than the carbon atom; thus, it is less polar than guanidinium group of arg . In all three mutations of arg138 with its analogues, the typical site for adp recognition still remains, and the purine moiety binds respectively, to oxy-, and sulfoguanidinium group . In the cases of cav138 and sarg138 mutations, total energies of the enzyme - substrate complexes decrease, while in the case of cav138 mutation, total energy increases . Fitness function value is almost the same when arg138 is replaced by cav, due to their structural similarity, and conformational changes in that case are very small (figures 7(a) and 7(b)). When arg138 is replaced by ncav and sarg, conformational changes in the binding site of the enzyme are bigger, and fitness function values are higher which is connected to the binding affinity of the substrate . The higher the fitness function value, the higher the affinity of the substrate to the enzyme and it will bind more strongly . This will decrease the rate of the enzymatic reaction and most probably will stop it in the case of ncan138 and sarg138 . There are some conformational changes and the values of fitness function indicate that, but newly formed enzyme - substrate complexes have total energies not very different from that of wild type enzyme - substrate complex . These mutations do not affect arg138 and the binding site for purine moiety remains unchanged (figure 7(c)). Exploring the sequence gagkg in the case of arg138 mutation, the biggest conformational changes occurred when arg138 is replaced by ncav (figure 8(a)). In all mutations in position 175, purine moiety could be recognized by the mutant enzyme, but because of the changes in the site responsible for interaction between two adp molecules, reaction could not be performed . From previously synthesized and biologically tested arginine analogues, three of them cav, ncav, and sarg could act as its antimetabolites . They could be recognized by arginylate - trna synthetase as substrates and could be included in numerous biologically important peptides and proteins . Once they become an element of proteins' sequence, for example, enzymes, they would cause crucial changes in their structure leading to loss of enzymatic action . As long as these compounds exhibit a prolonged biological effect, this most probably is due to their incorporation into important metabolic enzymes.
The lingual tonsils develop at 6.5 weeks between the second and third arches ventrally while palatine tonsils develop at 8 weeks from second pouch (ventral and dorsal). Tonsils are predominantly b - organs and b - lymphocytes comprise 5060% of tonsillar lymphocytes . Ample evidence shows that tonsils are involved in inducing secretory immunoglobulin production [4, 5]. Both adenoids and tonsils are favourably located to mediate immunologic protection of the upper aerodigestive tract as they are exposed to air borne antigens . Tonsils are particularly designed for direct transport of foreign material from the exterior to the lymphoid cells . Involution of the tonsils begins after puberty, resulting in a decrease of the b cell population and a relative increase in the ratio of t to b cells . The commonest indication for tonsillectomy is recurrent tonsillitis, which results in shedding the immunologically active cells and decreasing antigen transport function with subsequent replacement by stratified squamous epithelium [6, 7]. We investigated the common pathogens causing this condition in our hospital and report here our findings . Patients presenting at our clinic with signs and symptoms of chronic tonsillitis were enrolled for the study . The study was explained to them and where children were involved, to their parents . Before the operation began, the laboratory was informed and a technician stood by to collect the tonsil as soon as it was removed . Sterile wide - mouthed container was provided and the excised tonsils were aseptically put into them and carried immediately to the laboratory for processing . As soon as the tonsil reaches the laboratory, it is cut into two with a sterile surgical blade; the inner surfaces were swabbed with sterile cotton swab, and inoculated onto two blood agar plates, one macconkey agar and one chocolate agar plate . One blood agar plate was incubated anaerobically, the chocolate plate in 510% co2 while the rest of the plates were incubated aerobically . The aerobic plates and the co2 plate were examined after 24 hours; if no growth, they were reincubated for a further 24 hours after which if still no growth, they were discarded as negative . The anaerobic cultures were examined at 72 hours and if no growth they were reincubated for a total of 7 days . There were 34 bacterial isolates from 52 patients, giving a percentage positivity of 65.38% . Thirty isolates were gram - positive bacteria and only four were gram - negative, made up of two genera, klebsiella and pseudomonas . Staphylococcus aureus was the predominant isolate (15/34, 44.1%), followed by group b streptococcus (12/34, 35.3%). Others were, streptococcus pyogenes (group a streptococcus), 1/34, 2.94%; and untypable streptococcus spp . 2/34, 5.88% . The gram - negative bacteria consist of klebsiella pneumoniae 3/34, 8.82% and pseudomonas aeruginosa 1/34, 2.94% (table 1). There were no growths in 7 patients while 11 yielded growth of normal flora only . All the cases were chronic and most of them took antibiotics before presenting to us . The ratio of anaerobic to aerobic bacteria in saliva is approximately 10: 1 because of variations in oxygen concentration throughout the oral cavity . Invargsson et al . Revealed that streptococcus pneumoniae was recovered in 19% of healthy children, hemophilus influenzae in 13%, group a streptococcus in 5%, and moraxella (branhamella) catarrhalis in 36% . The frequency of pathogens decreases with age, possibly because of increased immunity . Because the oropharynx is colonized by many organisms, most infections of wadeyer's ring are polymicrobial . In our study, but in contrast with other researchers who found beta - hemolytic streptococci to be the predominant isolate [14, 15]. These authors also found that streptococcus pyogenes was isolated more frequently in recurrent tonsillitis while in the tonsillar hypertrophy, streptococci beta - hemolytic non a group predominated . It has been suggested that fine - needle aspiration can be used in identifying tonsil core bacteriology in clinical settings . Methicillin resistant staphylococcus aureus (mrsa), has been isolated from the surface and core tonsils in children . We did not encounter any mrsa in our study; and all the staphylococcus aureus isolated were sensitive to augmentin and vancomycin . It would appear from our results, that augmentin should be our drug of choice in future treatment of tonsillitis from this centre . The findings of kuhn et al . Supported the etiologic role of hemophilus influenzae and staphylococcus aureus in hypertrophic tonsils with or without inflammation [18, 19].
Pancreas is a doubled - entity organ, with both an exocrine and an endocrine component, reciprocally interacting and closely cooperating for the digestion, absorption, and metabolism of oral nutrients . Thanks to its lobulated tubulo - alveolar - acinar structure, endocrine islet cells secrete their hormones in the insulo - acinar portal vascular pathway, thus regulating and conditioning ductal and acinar cell exocrine activities . It has been recently demonstrated that the rise of insulin and other pancreatic islet peptides (amyline, c - peptide, and urocortin - iii), following the postprandial glucose rise, stimulates the exocrine pancreatic function . On the other hand, ductal and acinar cells, in turn, affect the physiology of endocrine islet cells through cytokines and growth factor secretion [2, 3]. Furthermore, the endocrine cells disseminated in the small bowel secrete two peptide hormones, glucagon - like peptide-1 (glp-1) and glucose - dependent insulinotropic hormone (gip), known as incretins, that are released soon after lipid and carbohydrate ingestion, thus stimulating a strong insulinic production . A deregulation of incretin secretion is one of the causes of the altered insulinic response in type ii diabetes . With regard to chronic pancreatitis patients, it is well described that they may suffer from a peculiar form of diabetes (type iii diabetes), characterized by the destruction of islet cells by inflammatory and fibrotic injury . Type iii diabetes differs from type i diabetes as the damage of pancreatic islets is not limited to insulin secreting beta cells but is more diffused as it also affects glucagon and pancreatic polypeptide secreting alpha and pp cells . In addition to this, in type iii diabetes the malabsorption of nutrients due to pancreatic exocrine insufficiency (pei) results in impaired incretin secretion with consequently diminished insulin release . Furthermore, as suggested by the 2012 pancreasfest recommendations, a quote of type iii diabetes patients may also have preexisting risk factors for type ii diabetes (e.g., insulin resistance, obesity, or dietary habits) that further complicate the optimal regulation of glucose metabolism resulting in a severe disease with large swings in blood sugar that are hardly controlled . On the other hand, less the prevalence of diabetes in the general population is much higher than that of chronic pancreatitis, this relationship is potentially of great clinical relevance . In the sixties, a few pioneering studies investigated exocrine pancreatic function in small series of diabetic patients, throughout direct duodenal juice collection (secretin cerulein test or sct) and reported changes in both output and hco3 concentrations [68]. More recently, thanks also to the availability of a noninvasive test for pancreatic exocrine function, such as measurement of fecal elastase, several studies aimed at investigating the prevalence of pei in large cohorts of diabetic patients . The present paper is aimed at reviewing findings of published studies investigating the prevalence of pei in diabetic patients and possible risk factors and mechanisms associated with its occurrence and severity . The cases of patients with type i and type ii diabetes will be discussed separately, in view of possible different features and pathogenic mechanisms . Abnormalities in histological features and imaging (mri, ct, and ultrasound) of the pancreas of diabetic patients have been reported, as well as atrophy, fibrosis, changes in size, and morphology; several and various hypotheses have been proposed to explain these phenomena implicated in pei occurrence in diabetics [9, 2225]. The damage of the exocrine cells in type i diabetes is most likely multifactorial, with a number of possible causes: (a) the lack of the trophic action of insulin (and possibly of glucagon and somatostatin) on acinar cells; (b) an involvement of the exocrine tissue in the autoimmune destruction of islet cells; (c) autonomic diabetic neuropathy leading to enteropancreatic reflex impairment; (d) hypoxic sufferance of exocrine tissue due to microvascular damage [26, 27]. In this view, type i diabetes, which is linked to a primary autoimmune process and is characterized by early occurrence, severe insulin deficiency, and long standing disease, with a high rate of neural and vascular complications, seems to be more frequently associated with pei than type ii . Pei seems to occur early in type i diabetes patients, and 2 studies assessing fecal elastase concentrations in children and young patients, ranging from 2 to 25 years of age, detected severe (defined by a cut - off of fecal elastase <100 g / g) to moderate pei (elastase <200 g / g) in 10% to 45% of screened subjects [28, 29]. In adult series of type i diabetes patients, the prevalence of both severe (1030%) and moderate (2256%) pei seems higher than in children, possibly suggesting that exocrine pancreatic function decreases in parallel with the duration of disease and the increase in insulin requirement . The largest study investigating risk factors for pei occurrence in 195 type i diabetes patients demonstrated a strong association between pei and disease duration, but these results were not confirmed in other settings and no association with insulin requirement or elevated hba1c was observed . However, these latter studies had several limitations, such as a smaller number of subjects included, not well - defined enrollment criteria, and poor investigation of risk factors for pei occurrence . Studies evaluating pancreatic exocrine function in adult type i diabetes patients are summarized in table 1 [915]. Physiopathological mechanism involved in pei occurrence in type ii diabetes seems to be similar to that reported above for type i diabetes . In particular, in these subjects, without autoimmune damage and insulin deficiency, autonomic neuropathy and microvascular damage may play a key role in inducing pancreatic atrophy and fibrosis . Hayden and colleagues observed that type ii diabetes human and rodent pancreas specimens show a loss of desmosomes and adherens junctions between islet and acinar cells, due to fibrosis and remodeling of the islet - acinar interface, that may result in an impaired function . Regarding pei prevalence in type ii diabetes more data are available, although these are somehow more heterogeneous . Studies evaluating pancreatic exocrine function in adult patients with type ii diabetes are summarized in table 2 [10, 12, 13, 1521]. In the largest prospective study, performed on 1231 diabetic patients, hardt and colleagues demonstrated a prevalence of pei of 35% in 697 patients with type ii diabetes . However, the observed correlations with disease duration and insulin therapy in the general population of 1231 subjects were not confirmed in the subgroup analysis of type ii diabetes patients . Notably, this study did not specifically exclude cases with previous history of pancreatic disease, thus leading to a possible bias . Subsequent studies, enrolling a smaller number of patients, confirmed a prevalence of pei of about 3040% in type ii diabetes . Although these results are often heterogeneous, most studies concord that the need of insulin therapy is associated with a higher prevalence and with the severity of pei [10, 13, 1521]. Early onset of type ii diabetes and long disease duration as well as poor glycemic control (expressed in particular as elevated hba1c) seem to be risk factors for the occurrence and severity of pei, although their role has not been confirmed in all studies and results were not always statistically significant [10, 13, 1521]. This supports the hypothesis that a long, complicated type ii diabetes, with a higher degree of microvascular damage, pancreatic fibrosis, and autonomic neuropathy, is more probably associated with pei occurrence . Interestingly, in most reports an elevated bmi seems to be a protective factor for the occurrence of pei occurrence . However, this is most likely a bias, as patients with severe pei would show malabsorption and malnutrition and consequent reduced bmi [10, 13, 15, 1721]. The reciprocal relationship between the endocrine and exocrine pancreatic function is an interesting and relatively poorly investigated area of research in which many questions still remain unanswered . Most studies investigating the prevalence of pei in both type i and type ii carry several limitations (summarized in tables 1 and 2). All these mentioned studies have a cross - sectional design, and they only evaluated the prevalence of pei and not its incidence over time in the course of the disease . One study prospectively evaluated pei with sct, although during a short and not standardized follow - up period and only in 17 type i diabetes patients . In contrast with previous observations, this latter study, with all the above mentioned limitations, observed no changes in the exocrine pancreatic function during the follow - up period . The reported prevalence of pei observed in type i (2574%) and type ii (2854%) diabetes in different studies assessing pei with fecal elastase measurement is also highly variable . This heterogeneous prevalence could be due to the low sensibility of fecal elastase, as reported by hahn and colleagues in a small series of type i diabetes patients, who underwent both direct and indirect pancreatic function tests . A recent study reported that a noninvasive 13c - mixed triglyceride breath test (c - mtgt) for evaluation of pancreatic exocrine function can detect mild to moderate pei in diabetes mellitus . However, the specificity of c - mtgt, compared to direct sct, is low in these patients, probably because nonpancreatic mechanisms contribute to decrease intestinal lipolysis . To better assess pei prevalence in diabetic patients, future studies should also take into account the dilution of elastase enzyme in stools during diarrhoea, which is, irrespectively to pei, a symptom often present in diabetic subjects because of bacterial overgrowth and diabetes - induced vascular or neuropathic complications [14, 31]. As regards the aetiology of pei, a long duration of disease, high insulin requirement, and poor glycemic control, expression of more severe disease seems to be associated with pei occurrence and severity . In type ii diabetes, a more severe form of disease is more frequently associated with systemic complications such as autonomic neuropathy and microvascular damage, determining fibrosis and atrophy of the pancreas, and loss of communication in the islet - acinar - ductal axis and gastroenteropancreatic system . In type i diabetes, the primary reduction of insulin levels due to the autoimmune mediated damage of islet cells results in a decreased trophic action on the exocrine cells . This combination of multiple factors may explain the observed higher prevalence of pei observed in type i compared to type ii diabetes (about 60% versus 30% of cases). Furthermore, it is unclear which diabetic patients would benefit from screening for pei in clinical practice, and there are very limited data regarding the possible impact of pancreatic enzyme replacement therapy (pert) on diabetic subjects with pei . In a small series of 8 patients affected by chronic pancreatitis with pei, half of them with type iii diabetes, knop et al . Evaluated the effect of pert on glucose, insulin, gip, and glp-1 profiles after meal . Interestingly, pert improved the response of gip, glp-1, and insulin response after meal, but not the postprandial glucose profile . One might, therefore, hypothesize that, also in diabetic patients with pei, pert might ameliorate the postprandial response of insulinotropic intestinal peptides and subsequently the insulin secretion . However, no studies have yet evaluated the impact of enzymatic replacement on insulin and incretin levels in response to meal, in these subjects . Only one prospective study evaluated the effect of pert on 39 insulin dependent diabetic (type i or ii) patients with pei (defined as fecal elastase i concentrations <100 g / g) compared to 41 similar patients receiving placebo . This study showed that pert improved steatorrhea and malabsorption symptoms in comparison to the controls receiving placebo but had no impact on glucose profile, insulin requirement, and hba1c levels . However, this study was performed on a small number of patients for a short period of time (3 months). Furthermore, all patients with non - insulin - dependent diabetes and with moderate pei (fecal elastase concentrations <200 g / g) were excluded, and insulin, glucagon, and incretin levels, that might be modified by the enzymatic treatment, were not assessed . Future studies evaluating the influence of pert on glycemic control and insulin requirement should enroll a larger and more homogeneous population of diabetic patients, with treatment enduring for a longer period of time, and should also possibly evaluate the relation between pert and incretins profile . On the other hand, the potential effect of treatment with incretins on the exocrine pancreas is also a controversial area for further research, as the relation between the exocrine and the endocrine pancreas represents a major field of investigation.
Seminal vesicle cysts are rarely diagnosed, but symptomatic congenital seminal vesicle cysts are reported in the literature describing zinner syndrome . We present the case of a 26-year - old patient admitted to the urology department because of abdominal pain . A left seminal vesicle cyst and left kidney agenesis were found on examination, and the patient qualified for laparoscopic removal of the left seminal vesicle cyst . The peritoneum was incised between the bladder and the rectum to reveal the left seminal vesicle, which was resected from the surrounding tissue . A tachosil (takeda pharmaceuticals, zurich, switzerland) hemostatic sponge was placed in the ledge after cutting the base of the seminal vesicle . The aim of this study was to evaluate usefulness of the laparoscopic technique to remove a cystic seminal vesicle . The patient was discharged from the hospital on the third postoperative day and remains symptom - free after 12 months . Histopathologic examination confirmed the presence of a seminal vesicle cyst, and renal agenesis and stenosis of the vas deferens suggests zinner syndrome . Symptomatic seminal vesicle cysts are a good indication for the application of a laparoscopic technique and, in our opinion, this is better than the open technique because of the view into the surgical area . Therefore, it should be used as the method of choice in treating seminal vesicle defects . Seminal vesicle cysts are a rarely diagnosed disease, seen in only 1 in 20,000 men . Most of these cysts coexist with a congenital syndrome, first described by zinner in 1914 . Zinner syndrome is characterized by the coexistence of vas deferens stenosis, renal agenesis, and an ectopic ureteral orifice in the prostatic part of the urethra or seminal vesicle . To date, only 100 cases of zinner syndrome have been described in the literature . Although the associated vesicle cysts are generally asymptomatic, they may cause symptoms such as perineal pain, painful ejaculation, hematospermia, abnormal micturition, lower urinary tract symptoms (luts), or dysuria . The suspected presence of a cyst is based on ultrasonography (usg) and is confirmed by transrectal ultrasonography (trus), computed tomography (ct), or magnetic resonance imaging (mri), which reveal fluid - filled spaces behind the bladder . In general, digital rectal examination demonstrates a large, soft, fluctuant pelvic mass in the region behind the prostate . Other methods, such as transrectal or transurethral puncture, aspiration, or transurethral resection of the seminal colliculus and vas deferens, carry the risk of recurrent cysts . Open surgery is not easy because of the location of the seminal vesicles deep beneath the bladder and the possibility of damage to the bladder and rectum . We describe a case of a seminal vesicle cyst in a patient with zinner syndrome who underwent laparoscopic surgery . A 26-year - old obese patient (body mass index of 36) was admitted to the urology department because of recurrent abdominal and perineal pain and dysuria . Usg revealed a fluid - filled space located behind the bladder on the left side . Ct and mri confirmed the presence of a left seminal vesicle cyst with a diameter of 8 cm (figures 1 and 2) and ipsilateral renal agenesis . The patient qualified for laparoscopic removal of the left seminal vesicle cyst because of persistent pain . The patient was placed in the trendelenburg position, and a catheter was placed into the urinary bladder and maintained during the whole procedure . Trocar 1 (10 mm) was added below the umbilicus for a 0-degree optic . Trocar 2 (5 mm) was placed for the left assistant hand to aid use of the suction, and trocar 3 (5 mm) was placed for the right assistant hand to use the retractor to hold the bowel or bladder; these were located between the umbilicus and the anterior iliac crest on the right side . Trocar 4 (10 mm) was for the right operator hand to use the harmonic scalpel or scissors, and trocar 5 (5 mm) was required for the left operator hand to use the bipolar dissector; these were placed between the umbilicus and the anterior iliac crest on the left side . Reaching the vesicorectal recess was difficult because of the patient's obesity and numerous peritoneal adhesions from a previous appendectomy . The first stage of treatment involved lifting the urinary bladder to reveal the vesicorectal recess . The peritoneum was then incised between the bladder and the rectum (figure 4). Cystic formations were located after removal of the fat layer (figure 5). This failed to separate the vas deferens, which was constricted and conjoined to the seminal vesicle as previously described in zinner syndrome . The seminal vesicle was excised using a harmonic scalpel and bipolar coagulation (figure 6). A hemostatic sponge (tachosil, takeda pharmaceuticals, zurich, switzerland) was placed in the ledge after cutting off the base of the seminal vesicle (figure 7). The excised cyst was placed in an endobag and removed from the body (figures 8 and 9). The patient was discharged home on the third postoperative day, and there were no postoperative complications . Histopathologic examination confirmed the presence of a seminal vesicle cyst . At the time of publication, the patient remains symptom free after 12 months of constant follow - up . A 26-year - old obese patient (body mass index of 36) was admitted to the urology department because of recurrent abdominal and perineal pain and dysuria . Usg revealed a fluid - filled space located behind the bladder on the left side . Ct and mri confirmed the presence of a left seminal vesicle cyst with a diameter of 8 cm (figures 1 and 2) and ipsilateral renal agenesis . The patient qualified for laparoscopic removal of the left seminal vesicle cyst because of persistent pain . The patient was placed in the trendelenburg position, and a catheter was placed into the urinary bladder and maintained during the whole procedure . Trocar 1 (10 mm) was added below the umbilicus for a 0-degree optic . Trocar 2 (5 mm) was placed for the left assistant hand to aid use of the suction, and trocar 3 (5 mm) was placed for the right assistant hand to use the retractor to hold the bowel or bladder; these were located between the umbilicus and the anterior iliac crest on the right side . Trocar 4 (10 mm) was for the right operator hand to use the harmonic scalpel or scissors, and trocar 5 (5 mm) was required for the left operator hand to use the bipolar dissector; these were placed between the umbilicus and the anterior iliac crest on the left side . Reaching the vesicorectal recess was difficult because of the patient's obesity and numerous peritoneal adhesions from a previous appendectomy . The first stage of treatment involved lifting the urinary bladder to reveal the vesicorectal recess . The peritoneum was then incised between the bladder and the rectum (figure 4). This failed to separate the vas deferens, which was constricted and conjoined to the seminal vesicle as previously described in zinner syndrome . The seminal vesicle was excised using a harmonic scalpel and bipolar coagulation (figure 6). A hemostatic sponge (tachosil, takeda pharmaceuticals, zurich, switzerland) was placed in the ledge after cutting off the base of the seminal vesicle (figure 7). The excised cyst was placed in an endobag and removed from the body (figures 8 and 9). The patient was discharged home on the third postoperative day, and there were no postoperative complications . Histopathologic examination confirmed the presence of a seminal vesicle cyst . At the time of publication, the patient remains symptom free after 12 months of constant follow - up . Zinner syndrome is a rarely diagnosed disease characterized by renal agenesis, an ectopic ureteral orifice in the prostatic part of the urethra or seminal vesicle, vas deferens stenosis, and a seminal vesicle cyst . Seminal vesicle cysts are generally small (<5 cm), asymptomatic, and do not require treatment; however, they can also achieve a much greater size . A growing cyst may result in perineal pain, painful ejaculation, hematospermia, abnormal urination, luts or dysuria, and infertility . Digital rectal examination reveals a resilient formation palpable behind the prostate, but only usg is capable of diagnosing a cyst and determining its relative size, location, or structure . The lesion is anechoic, has a smooth wall, and is filled with homogeneous fluid . Additional diagnostic methods such as transrectral ultrasonography, ct, or mri are used to exclude other pelvic lesions, as well as to find the causes of cyst formation and confirm the nature of a cyst . We used ct and mri to identify a smooth - walled anechoic space, including the seminal vesicle, located behind the bladder . The image was so characteristic that the diagnosis of zinner syndrome was beyond doubt, especially given the concurrent presence of ipsilateral renal agenesis . Cystoscopy can also help to confirm the absence of a ureteral orifice . In the patient described here, usg demonstrated a fluid - filled space behind the bladder and the absence of the left kidney . Initially, the patient was treated conservatively because this can sometimes elicit the desired effect . One case of successful conservative treatment of a patient with luts symptoms for zinner syndrome has been described . They received short - term antibiotic treatment and -blockers for 6 months, and the symptoms were resolved permanently after these treatments . In our patient, the predominant symptom was severe perineal and abdominal pain, which has been reported as the most common indication for surgical treatment . The patient was obese, which was an indication for this treatment . However, reported poor effects of therapy with a high probability of cyst recurrence and the exposure of a young patient to the possible need to repeat the procedure prompted us to attempt to remove the cyst laparoscopically . We performed the operation in a similar way to the first stage of laparoscopic radical prostatectomy with transperitoneal access, as performed in our institution between 1999 and 2002 . In accordance with the original montsouris technique, in the first stage of the operation we always prepared the seminal vesicles, reaching them through an incision in the peritoneal vesicorectal recess . Similarly, we inserted 5 trocars (3 10 mm and 2 5 mm) in the typical locations for prostate resection . Although numerous adhesions and a thick layer of body fat complicated the surgery, the view of the operative field was ideal after reaching the vesicorectal recess . We clearly saw the cystically expanded left seminal vesicle and resected it from the surrounding tissue . Seo et al described 4 operations involving laparoscopic removal of seminal vesicle cysts that used 4 trocars . Treatment began by preparing the retroperitoneal space to identify the kidney and ureter and moving downward to reach the seminal vesicle . However, jang et al reported the successful application of laparoendoscopic single - site surgery, and, as we did, they also began preparation by making an incision of the peritoneum in the vesicorectal recess to directly reach the seminal vesicle . The literature also describes the use of a robot to remove seminal vesicle cysts with easier instrument manipulation, greater movement precision, and excellent visualization . The operation reported here lasted 3 hours, which was slightly longer than the surgeries described by other authors (from 100 to 180 minutes). However, our patient was very obese and had numerous peritoneal adhesions from a previous complicated appendectomy, and this prolonged the time needed to reach the recess . The preparation of the seminal vesicle using a laparoscopic technique was not difficult; a harmonic scalpel facilitated preparation, and placing a tachosil (takeda pharmaceuticals, zurich, switzerland) hemostatic sponge allowed us to control bleeding from numerous small vessels . The view into the operative field achieved with the laparoscopic technique was perfect and certainly much better than with the classical treatment; this point is emphasized by many who use this surgical technique . The patient was discharged home on the third postoperative day, and we did not record any intra- or postoperative complications . Rather, the long hospital stay after laparoscopic surgery was a benefit of the health care system in poland . A good view of the operative field with appropriate magnification makes the procedure safer, which is extremely important in such a difficult and inconvenient location in proximity to the rectum and bladder . It is difficult to draw general conclusions based on one case, but, as other authors have done, we emphasize that the laparoscopic or robotic technique is an excellent choice for treating seminal vesicle diseases . The diagnosis of a seminal vesicle cyst with renal agenesis and stenosis of the vas deferens is suggestive of the very rare condition of zinner syndrome . Compared with the open technique, laparoscopic surgery is safe and, in our opinion, provides a better view of the operated area.
Approximately 225,000 primary total hip arthroplasties (thas) are performed in the united states per year, and projections indicate that more than 4 million thas will be performed annually by 2030.1 neurologic injury after tha is reported to occur in 0.09 to 3.7% of primary thas and 0 to 7.4% of revision thas.2 noted causes of neurologic injury include direct intraoperative nerve injury, leg lengthening, excess retraction, cement extravasation, cement - related thermal damage, patient positioning, and postoperative hematoma.2 the majority of these injuries are symptomatic within the early postoperative period and can be managed conservatively with close follow - up . Delayed onset of neurologic injury we report a case of neuropathy caused by malposition of an acetabular cup screw causing direct injury to the sciatic nerve . The patient developed progressively worsening loss of ankle dorsiflexion, resulting in complete footdrop 2 years after initial tha . The presentation, intervention, and outcome of other cases of delayed neuropathy are reviewed in the present report . Delayed neuropathies are most often caused by irritation from hardware, component failure, or wear - related pseudotumors . The patient was a 52-year - old woman with end - stage arthritis of her bilateral hips with previous uncomplicated right tha that underwent an elective left tha . No intraoperative complications, abnormal anatomic landmarks, or events were noted at the time of surgery . The patient's initial orthopedic postoperative course was complicated by paresthesias of the right lower extremity, severe hypesthetic pain primarily in the lateral and anterior aspect of the right calf, and severe sciatica . During her hospital stay, the patient's sensation and pain improved; however, less than 1 year after her tha she noted gradual onset of weakness in her right lower extremity ., she had developed a complete footdrop with loss of ankle dorsiflexion and required the use of an ankle - foot orthosis (afo) to ambulate . She underwent an emg at an outside facility, had a neurologic evaluation, and was undergoing physical therapy . The patient had 0/5 dorsiflexion strength with 3/5 eversion and 5/5 inversion and plantarflexion strength . Decreased sensation was noted in the lateral aspect of the leg and the dorsum of the foot . She had no gross deformity of the lower extremity and had normal quadriceps and hamstring strength at the knee and the hip . Emg showed severely reduced amplitude of the peroneal compound muscle action potentials and unrecordable superficial peroneal sensory response . Fibrillation potentials were seen in the peroneal division of the sciatic nerve with minimal evidence of reinnervation changes to the muscle units . This localized the lesion to proximal to the short head of the bicep femoris muscle . This led to obtaining advanced imaging consisting of a computed tomography (ct) of the pelvis, which demonstrated a malpositioned acetabular cup screw within the soft tissues adjacent to the sciatic nerve (fig . The sciatic nerve was dissected via internal neurolysis into peroneal and tibial divisions at the level of the buttock . The division of the sciatic complex was dissected proximally to the exit of the sciatic from the pelvis . The peroneal portion of the nerve was found to be compromised by the malpositioned screw . There was no neuroma identified at the site of nerve compression, although there was evidence of nerve displacement by the protruding screw . The nerve was stimulated locally and nerve action potentials were recorded proximal and distal to the site of injury . Both peroneal and tibial portions of the nerve were tested separately to avoid false - positive conduction from the tibial division . The intraoperative recordings showed preservation of conduction across the site of nerve impingement (fig . The nerve was reflected off the head of the screw and gently moved proximally toward the sciatic notch . When the nerve was free of the screw and protected, a titanium cutting drill bit was used to shorten the screw so that it was flush with the bony interface . Repeated stimulation of the nerve confirmed that action potentials were still able to propagate past the site of injury . The patient is now 2 years out of surgery and has demonstrated some recovery of peroneal nerve function, supporting the decision to pursue external neurolysis . The patient's improvement has not been complete and the patient requires an external orthosis . The patient was a 52-year - old woman with end - stage arthritis of her bilateral hips with previous uncomplicated right tha that underwent an elective left tha . No intraoperative complications, abnormal anatomic landmarks, or events were noted at the time of surgery . The patient's initial orthopedic postoperative course was complicated by paresthesias of the right lower extremity, severe hypesthetic pain primarily in the lateral and anterior aspect of the right calf, and severe sciatica . During her hospital stay, the patient's sensation and pain improved; however, less than 1 year after her tha she noted gradual onset of weakness in her right lower extremity ., she had developed a complete footdrop with loss of ankle dorsiflexion and required the use of an ankle - foot orthosis (afo) to ambulate . She underwent an emg at an outside facility, had a neurologic evaluation, and was undergoing physical therapy . The patient had 0/5 dorsiflexion strength with 3/5 eversion and 5/5 inversion and plantarflexion strength . Decreased sensation was noted in the lateral aspect of the leg and the dorsum of the foot . She had no gross deformity of the lower extremity and had normal quadriceps and hamstring strength at the knee and the hip . Emg showed severely reduced amplitude of the peroneal compound muscle action potentials and unrecordable superficial peroneal sensory response . Fibrillation potentials were seen in the peroneal division of the sciatic nerve with minimal evidence of reinnervation changes to the muscle units . This localized the lesion to proximal to the short head of the bicep femoris muscle . This led to obtaining advanced imaging consisting of a computed tomography (ct) of the pelvis, which demonstrated a malpositioned acetabular cup screw within the soft tissues adjacent to the sciatic nerve (fig . The sciatic nerve was dissected via internal neurolysis into peroneal and tibial divisions at the level of the buttock . The division of the sciatic complex was dissected proximally to the exit of the sciatic from the pelvis . The peroneal portion of the nerve was found to be compromised by the malpositioned screw . There was no neuroma identified at the site of nerve compression, although there was evidence of nerve displacement by the protruding screw . The nerve was stimulated locally and nerve action potentials were recorded proximal and distal to the site of injury . Both peroneal and tibial portions of the nerve were tested separately to avoid false - positive conduction from the tibial division . The intraoperative recordings showed preservation of conduction across the site of nerve impingement (fig . The nerve was reflected off the head of the screw and gently moved proximally toward the sciatic notch . When the nerve was free of the screw and protected, a titanium cutting drill bit was used to shorten the screw so that it was flush with the bony interface . Repeated stimulation of the nerve confirmed that action potentials were still able to propagate past the site of injury . The patient is now 2 years out of surgery and has demonstrated some recovery of peroneal nerve function, supporting the decision to pursue external neurolysis . The patient's improvement has not been complete and the patient requires an external orthosis . Tha is a widely used procedure for the treatment of end - stage hip arthritis that is able to restore functional ability to hundreds of thousands of patients a year in the united states . There is a known risk of immediate neuropathy from mechanisms such as intraoperative compression from retractors, hematoma, limb lengthening, and inappropriate patient positioning . However, there is also a rarer risk of delayed neuropathy after tha that has different causative pathologies . Our findings from the literature suggest that late complications from tha may be more likely due to migration of hardware over time with subsequent nerve compression or reactive mass or fluid collection . In our case, a long, malpositioned screw was found in the soft tissue compressing the peroneal branch of the sciatic nerve . A search of the pubmed database was conducted using the search term delayed neuropathy after total hip arthroplasty covering the years 1950 through 2015 . Fifteen case reports on 16 patients were selected based on involvement of a neuropathy that appeared after tha.3 4 5 6 7 8 9 10 11 12 patients presenting prior to 1 month included those that developed postoperative hematomas, had poor patient positioning, had previously undiagnosed hereditary neuropathy with liability to pressure palsies (hnpps), or had anesthetic - induced inflammatory neuropathy and were felt to qualify as early postoperative complications . Delayed onset was defined as those with neuropathies developing over 1 month from surgery . Abbreviations: afo, ankle - foot orthosis; ct, computed tomography; emg, electromyography; ir, interventional radiology; mri, magnetic resonance imaging; tha, total hip arthroplasty . Date of presentation ranged from 4 months to 9 years postoperatively . Presenting symptoms included eight patients with pain, nine with sensory deficits, eight with motor deficits, and four with loss of reflexes . Regarding patient workup, three received hip radiographs, four had cts, two had magnetic resonance imaging (mris), and nine underwent emgs . Ten patients received operative intervention, whereas one was treated conservatively with a brace and one with no intervention . Mechanisms of injury include three patients with mass formation adjacent to the construct, one patient with limb - lengthening injury, and seven patients with hardware migration and compression . Of those with mass formation next to the construct, one was felt to be inflammatory pseudotumor whereas two were felt to be wear - related cysts caused by metal debris . After intervention, four patients made a full recovery of function, whereas six out of eight patients with preintervention motor deficits had persistent motor deficits and one without preintervention motor deficits developed a foot drop postoperatively . Out of the nine patients with sensory deficits, four had persistent sensory loss after intervention . Patients with delayed neuropathy varied in presentation with pain, sensory, and motor symptoms . Most patients were evaluated with an emg that can objectively quantify degree of motor compromise and aid in lesion localization . As the causes of delayed nerve injury are largely local anatomical changes, ct and mri can be helpful in identifying the cause of neuropathy . Emg localization of the level of deficit, combined with screw malposition, suggested that it was the cause of the patient's sensory and motor loss . Delayed neuropathy is a rare complication after primary tha that can be caused by hardware irritation as in the present case . Patients can present with pain, sensory loss, motor loss, and decreased reflexes that are best worked up with emg to localize the area of neuropathy and imaging to identify the root source . Results from the literature show that operative intervention is often pursued to explore and directly visualize the nerve with limited results in the literature showing modest relief of pain and sensory symptoms and poor restoration of motor function.
Population ageing is a global phenomenon . In the mid-21st century, the number of older persons was 202 million which consisted of 8% in the whole population, but it reached about 841 million which is 4 times higher than that in 1950 . Fukuoka et al . (2016) predicted that the number of the older persons will triple by 2,050 to attain 2 billion, reaching 21% of world population . South korea is one of the great examples of the phenomenon . This country has shown the fastest growing rate of the ageing population in the world . For instance, while other advanced countries such as france, the united states, and japan have taken 115 yr, 73 yr and 24 yr, respectively, korea has taken 18 yr to enter the aged society (ahn et al ., 2014). In addition, the proportion of population who are over 65 yr in korea will be reached to be nearly 37% by 2050 (choi et al ., 2016). The dramatic increase of the numbers of older persons in the population of korea has brought about a variety of social issues such as physical and mental health, social relationship, and financial problem . Of those issues, most elderly people face various and serious health problems which can hugely affect their lives (spar and la rue, 2002). Indeed, health problems in the elderly can cause enormous economic damage at the level of the government and can lead to the huge losses of their autonomy and independence at the level of the individual . Therefore, health is the most important issue not only for the elderly, but for the government . Campos (2011) argues that it is difficult to define or discuss health without mention of wellness because the two terms are closely interwoven . In the words, the definition of health includes wellness which implies a sense of welling in all aspects of life . (2006) define wellness as: a lifelong process that at any given time produces a positive state of personal well - being, of feeling good about yourself; of optimal physical, psychological, and social functioning; and the control and minimization of both internal and external risk factors for both diseases and negative health conditions . It suggests that what you believe, feel and do have an influence on your health . In the same vein, hoeger and hoeger (2007) assert that wellness implies a constant and deliberate effort to stay healthy and achieve the highest potential for well - being . Based on both definitions, kim (2000) classifies wellness into five dimensions: (a) physical wellness can be defined as the ability to carry out daily tasks through physical activity and proper nutrition; (b) social wellness can be the ability to build healthy, nurturing and supportive relationships and to foster a strong connection with others; (c) spiritual wellness can be characterized as a guiding sense of meaning or value in life; (d) intellectual wellness can be the ability to learn knowledge and skills and apply them to daily tasks; and (e) emotional wellness can be defined as the ability to handle emotions and express them appropriately and comfortably . The improvement of wellness for the elderly may play an important role in increasing overall quality of life (liao and brunner, 2016). For the elderly, however, aging may lead to gradual loss of physical, mental, social and intellectual functions and in turn sport and physical activity has been recognized as an important agent in improving wellness in the elderly (kim and lee, 2011; stephenson et al ., 2007). Considering the positive effects of physical activities in wellness for the elderly, in this study researcher introduces a new form of exercise known as pilates developed by joseph pilates in the early 20th century that contributes to improving the balance, muscle strength and coordination . Pilates emphasizes the mind - body connection and it is also getting popular in korea . Moreover, it is not necessary to have the open space to practice it (mokhtari et al . The exercise can be performed on a mat or with equipments such as the reformer, cadillac, and chair (roh, 2015). To date, previous research has focused on its positive role for youth and adults (cromwell et al ., 2007; johnson et al ., 2007). However, there has been little research on how a pilates exercise affects on the elderly s wellness . Therefore, the purpose of this study is to examine the effect of a 12-week pilates intervention on wellness of the elderly . The participants of this research were sampled from the elderly (aging over 65 yr old) who participated in continuing education centers located in incheon metropolitan city . Researcher explained the purpose of the study and 93 elderly agreed to participate in pilates exercise in the beginning of this study, but 5 participants dropped out due to personal reasons such as health issue (4) and the lack of time (1). As a result, a total of 88 participants (63 females: 68.574.43 yr, 26 males: 68.84.69 yr) completed a 12-week pilates intervention for this research . All participants received a pilates exercise training, 3 sessions of 50 min per week for a duration of 12 weeks . Pilates exercises which were used in this study were based on the programs that were used in previous studies (kaesler et al . The exercises were performed into two parts . For the first six weeks, pilates on the mats was performed and the second part was performed with bands for the rest six weeks . This questionnaire was composed of 23 items; 4 items in background information and 19 items in wellness . Before and after performing the exercises, all participants completed a questionnaire to measure the efficiency of pilates exercises on wellness . Wellness scale was developed by kim (2000) with 19 questions consisted of five subvariables such as physical (5 items), social (4 items), spiritual (3 items), intellectual (3 items), and emotional wellness (4 items). The responses to all items were made by the likert scale with 1 point in not at all and 5 point in strongly agree . To test the validity of the questionnaire, a meeting of a panel of experts was held with 3 professors in a realm of socio - psychology . A principle component analysis was conducted to minimize the number of factors and varimax rotation was used as a rotation method . The reliability coefficient ranged from 0.708 to 0.847 which was judged to be very reliable . 20.0 (ibm co., armonk, ny, usa) was used to analyze all data collected in this study . First, an exploratory factor analysis and a reliability analysis were conducted to verify the validity and reliability of the questionnaire . Second, the paired t - test was conducted to verify the difference between pre- and posttest . The significance level of () the participants of this research were sampled from the elderly (aging over 65 yr old) who participated in continuing education centers located in incheon metropolitan city . Researcher explained the purpose of the study and 93 elderly agreed to participate in pilates exercise in the beginning of this study, but 5 participants dropped out due to personal reasons such as health issue (4) and the lack of time (1). As a result, a total of 88 participants (63 females: 68.574.43 yr, 26 males: 68.84.69 yr) completed a 12-week pilates intervention for this research . All participants received a pilates exercise training, 3 sessions of 50 min per week for a duration of 12 weeks . Pilates exercises which were used in this study were based on the programs that were used in previous studies (kaesler et al . For the first six weeks, pilates on the mats was performed and the second part was performed with bands for the rest six weeks . This questionnaire was composed of 23 items; 4 items in background information and 19 items in wellness . Before and after performing the exercises, all participants completed a questionnaire to measure the efficiency of pilates exercises on wellness . Wellness scale was developed by kim (2000) with 19 questions consisted of five subvariables such as physical (5 items), social (4 items), spiritual (3 items), intellectual (3 items), and emotional wellness (4 items). The responses to all items were made by the likert scale with 1 point in not at all and 5 point in strongly agree . To test the validity of the questionnaire, a meeting of a panel of experts was held with 3 professors in a realm of socio - psychology . A principle component analysis was conducted to minimize the number of factors and varimax rotation was used as a rotation method . A reliability coefficient which represents internal consistency the reliability coefficient ranged from 0.708 to 0.847 which was judged to be very reliable . 20.0 (ibm co., armonk, ny, usa) was used to analyze all data collected in this study . First, an exploratory factor analysis and a reliability analysis were conducted to verify the validity and reliability of the questionnaire . Second, the paired t - test was conducted to verify the difference between pre- and posttest . The significance level of () the collected data went through descriptive statistics such as mean and standard deviation and paired t - test was used to determine whether the mean difference between pre- and posttest . There were statistically significant differences in physical (t=2.762, p<0.01), social (t=3.362, p<0.001), spiritual (t=2.307, p<0.05) and emotional wellness (t=2.489, p<0.05) for the elderly between pre- and post - pilates intervention . This research examined the effect of a 12-week pilates exercise on wellness in elderly women . Findings of the study revealed that pilates exercises might be a useful tool for helping older persons to improve various dimensions of wellness . In other words, pilates exercisesprogram for 12 weeks significantly improved elderly s wellness such as physical, social, spiritual and emotional wellness . After reviewing the literature, it was found that the pilates exercise contributes to improving physical wellness in the elderly (hall et al ., 1999; (2015), which studied on the effects of pilates exercise training on physical fitness and welling in the elderly, argue that pilates exercise can be considered a proper physical activity for the elderly . Consequently, this exercise increases physical fitness and in turn it improves overall quality of life . Previous studies also have shown that pilates exercise reinforces emotional wellness in older persons (siqueira rodrigues et al ., 2010). 2015) investigated the effect of 12-week pilates exercises of older adults who completed for duration of 12 weeks . The results revealed that pilates exercises improve aspects of the health - related quality of life of older adults . In addition, kim and lee (2011) investigated the effect of leisure sports on wellness in the elderly . Older persons who spent more time in active exercises build a better relationship with others and have interpersonal relation skills . The more they participated in physical activities, the more they are socially healthy . In this study, therefore, pilates exercises may play an important role in improving overall wellness in the elderly and in turn it may affects older persons overall health and wellbeing . This research focuses primarily on the effect of a 12-week pilates intervention on wellness in the elderly . In other words, therefore, future research should be designed with a control group such as other exercise group or nonexercise group.
Tuberculosis remains the most important infectious disease globally, and mycobacterium tuberculosis is thought to be present in one - third of the world's population with 810 million new cases of active tuberculosis occurring annually worldwide . In 2013, an estimated 9.0 million people developed tuberculosis, and 1.5 million died from the disease . Almost all cases of tuberculosis are caused by m. tuberculosis, with m. bovis contributing less than 1.4% of all pulmonary cases outside of africa and ~2.8% of cases in africa with a crude incidence of 7 cases per 100,000 population . Because of the difficulties of chemotherapy, the incidence of multidrug - resistant strains of m. tuberculosis has increased in many areas during recent decades . This deterioration in the global situation highlights the need for new therapeutic agents against mycobacterial diseases . Recent research has shown that defensins have bactericidal activity against m. tuberculosis and indicate their potential to play a more significant role in tuberculosis control than what was previously considered . Protective immunity against mycobacterial infections . However, an efficient innate immune response may also be important in natural resistance against mycobacterial infection in addition to maintaining longer - term control of bacillary growth during latent infection . Alveolar macrophages and lung epithelial cells are the main cells to first encounter m. tuberculosis during primary infection . Studies of human airway epithelia in vivo or in vitro show that they can generate antimicrobial activity through the production of antimicrobial peptides [5, 6]. In addition, defensins account for a high proportion of the antibacterial activity associated with neutrophil granules [7, 8]. Defensins act as a bridge linking innate and acquired immunity largely through their chemotactic properties . They belong to a family of small (35 kda) cationic cytotoxic and oxygen - independent peptides that are active against a wide spectrum of microorganisms including bacteria, viruses, and fungi . This review presents a brief summary of their role in immunity with specific reference to human and animal tuberculosis and explores their potential as a novel approach to therapy or prophylaxis . There are three major classes of defensins expressed by different cells within the vertebrate world; -, -, and -defensins are differentiated by their structure and antimicrobial activity . -defensins, which have been identified in humans, monkeys, and several rodent species, are particularly abundant in polymorphonuclear neutrophils (pmns), certain macrophage populations, and paneth cells of the small intestine [8, 10, 11]. Human neutrophil -defensins (hnps) 14 constitute ~30% of proteins in the azurophilic granules of pmns . However, against gram - positive bacteria, hnp-1, hnp-2, and hnp-3 account for most of the total defensin content and have greater antimicrobial activity than hnp-4 . In contrast, the potency of hnp-4 against gram - negative bacteria is greater than those of hnp-1 and hnp-2 . -defensins 5 and 6 are found in paneth cells, the epithelial granulocytes of the small intestine [14, 15]. -defensins show activity in vitro against gram - negative and gram - positive bacteria and fungi that can be modulated by environmental conditions such as redox and ph . In contrast to -defensins, -defensins are largely expressed in epithelial tissues [18, 19]. The first -defensin was identified in bovine tongue tissue, and -defensins subfamilies have now been reported in primates (humans and apes), bovines, and rodents such as rats and mice [6, 20]. Furthermore, pigs and other farm animals including birds express only -defensins [2022]. As the most comprehensively studied, -defensins possess the widest taxonomic distribution, including invertebrates and plants, indicating an ancient point of origin . Human -defensin 1 (hbd-1) is expressed constitutively by a number of body systems including the urogenital tract and respiratory tract [24, 25]. Hbd-2 was discovered in extracts of lesional scales from patients suffering from psoriasis and is expressed by inflamed skin, lung, oral mucosa, and ocular surfaces . Its expression by epithelial cells can be induced by tnf- and interleukin- (il-) 1 but also by bacteria . Hbd-3 is expressed mainly by the skin and tonsils, and hbd-4 is expressed by many tissues but is particularly highly expressed in the gastric antrum and testes . The expression of hbd-1 is constitutive, whereas hbd-24 are inducible and are thought to play a crucial role against bacterial infection as part of the epithelial barrier . Proinflammatory cytokines, bacteria, and fungi have all been found to increase the expression of these defensins in cultured keratinocytes . -defensins are the only defensin subfamily with a circular structure, which likely originated through mutation of a preexisting -defensin gene in old world monkeys . They were first found in the monocytes and neutrophils of the rhesus monkey (macaca mulatta) and are the least numerous subfamily of mammalian defensins, with only three -defensins recognized (rhesus theta defensin (rtd) 13) from studies within leukocytes . Circular defensin isoforms also exist with five identified in peripheral blood leukocytes and four in the bone marrow in the olive baboon (papio anubis). Less is known about the distribution and diversity of -defensins because only 11 different -defensins have been isolated from three species of primates since their discovery in 1999 . The antimicrobial activity of -defensins is thought to be their natural function, and they bind to and neutralize bacterial toxins [36, 37]. For example, the human -defensin retrocyclin-1 and its analogues are active against c. albicans and l. monocytogenes and bind to anthrax lethal toxin . To date, more than 17 human defensins have been reported . However, many more -defensins have been reported by in silico analysis, and a genome - wide computational search has identified more than 30 human uncharacterized -defensin genes with full biological significances currently undetermined . The role of defensin chemotactic activity in initiating and regulating the immune response is now well known [3032]. They possess chemotactic activities for immature memory t - cells and dendritic cells through the chemokine receptor ccr6 . Hbd-2 and hbd-3 can combine with both cpg and host dna to form aggregates that resemble dna nets, which may enhance the intracellular uptake of cpg and self dna and activate plasmacytoid dendritic cells (pdcs) to promote dna - induced ifn- production in a toll - like receptor 9- (tlr9-) dependent manner . Subcutaneous injections of these complexes showed enhanced infiltration of inflammatory cells at the injection site, indicating a potential pathophysiological role for defensin / dna complexes in contributing to inflammation . A recent study showed that murine -defensin 2 (mbd-2) immunostaining in tuberculous mice was essentially localized to cells with dendritic morphology located near the mediastinal lymph nodes and showed a high level of gene expression . This suggests that -defensins may play an important role in the initiation of a th1 response as a link between the innate and adaptive immune responses . Hnps can also promote b- and t - cell interactions by modulating the th1- and th2-type cytokines [43, 44]. The expression and production of defensins are activated by a number of routes including direct recognition of pathogen - associated molecular patterns (pamps), such as lps, by tlr . This initiates mapk- or nf-b - dependent cascades that culminate in a proinflammatory response involving the secretion of cytokines, chemokines, and defensins, which themselves also have the capacity to induce defensin secretion . Thus, infection of cells with live mycobacteria leads to the induction of tnf and hbd-2 . Defensins are also thought to contribute to the inflammatory processes by inducing histamine release by mast cells and increasing hyperresponsiveness of the airways to histamine . Besides their antimicrobial and potential proinflammatory activities, defensins also display anti - inflammatory roles by binding to c1q to inhibit activation of the classical pathway of complement activation and have been demonstrated to inhibit fibrinolysis . Some defensins are also able to limit the inhibitory action of glucocorticoids on the suppressor functions of t - lymphocytes, which was abolished after adrenalectomy . The main mode of antibacterial action is the direct lysis of microorganisms through permeabilization of cell membranes either whilst the bacteria are extracellular or after phagocytosis . It is believed that electrostatic binding between the arginine groups of cationic defensins and membranes rich in anionic phospholipids induces the formation of voltage - regulated channels, which causes the leakage of intracellular metabolites . Some defensins can also bind avidly to membrane glycoproteins, which may be in part responsible for their antiviral activity . This is supported by the fact that defensins do not have antiviral activity against nonenveloped viruses . In addition, defensins can bind to polyanionic molecules such as dna via electrostatic interaction after entering the bacterial cell . Since hnps also target genomic dna, inducing single - strand dna breaks, it has been hypothesized that adenosine 5-diphosphate ribosylation might play a regulatory role in the biological properties of arginine - rich hnps . Defensins are highly antibacterial even in micromolar concentrations for both gram - negative and gram - positive bacteria, including mycobacteria . Pathogens that colonize sites where defensins may be present in high concentrations have developed mechanisms to resist their antibacterial activity . A number of genes responsible for defensin resistance have been identified in different pathogens . A staphylococcal gene, mprf, confers resistance to several antimicrobial peptides including defensins and related genes and has been found in the genomes of several other pathogens such as m. tuberculosis, pseudomonas aeruginosa, and enterococcus faecalis . Mprf reduces the negative charge of the membrane surface and leads to decreased binding of the cationic defensins by modifying phosphatidylglycerol with l - lysine in the membrane lipids . The phop gene in salmonella enterica serovar typhimurium is also thought to contribute to defensin resistance because a phop mutant shows significantly greater sensitivity to defensins . Potential clinical application of defensins must thus be reviewed in the light of possible development or acquisition of resistance . However, their nonspecific mode of action suggests that they should show promise in averting the development of resistance . Moreover, studies have demonstrated that resistance is less frequent than that observed for conventional antibiotics [5961], and selections for resistance in susceptible strains of m. tuberculosis, pseudomonas aeruginosa, and enterococcus faecalis have failed . In addition to their toxic effects on microorganisms, there are a number of reports indicating cytotoxicity for eukaryotic cells . High concentrations of hnps are found in the airway secretions of patients with chronic inflammatory lung disorders . It has been reported that hnps are cytotoxic to airway epithelial cells and can induce chemokine secretion in several cell types such as macrophages . Other studies showed that hnps are cytotoxic not only against various kinds of human and murine tumor cells but also against a wide range of normal cells, including human endothelial cells, lymphocytes, murine thymocytes, pmns, and spleen cells in a concentration - dependent (25100 g / ml) and time - dependent manner [62, 64]. As cytotoxic molecules, hnps can cooperate with hydrogen peroxide, which is also secreted by activated neutrophils, to affect synergistic cytolytic activity in vitro . This interaction may contribute to granulocyte - induced cytotoxicity in vivo [59, 65]. Some studies showed that the minimum inhibitory concentration and median inhibitory concentration of hnp-1 were 2.5 g / ml and 0.8 g / ml, respectively, which are much lower than the harmful concentrations to normal cells . This indicates that defensins have a relatively low level of cytotoxicity to normal cells at antimicrobicidal concentrations . Low cytotoxicity in vitro might be due to the presence of fetal bovine serum in the culture media, as serum proteins can protect mammalian target cells . The inhibitory effect of fetal calf serum (fcs) on lysis and binding can be completely accounted for by its content of albumin . Not only could fcs prevent defensin binding, but also it removed membrane - bound defensin molecules from the targets . Several proteins that can bind to defensins have been identified, and some of them may work as defensin carriers for clearance from tissues and blood . A high concentration of -defensins has been detected in bronchoalveolar lavage (bal) samples and pleural fluid from patients suffering from pulmonary tuberculosis, and significant levels of -defensins have been detected in bronchoalveolar lavage fluid from patients with m. avium - intracellulare infection . Moreover, studies of gene expression profiles by microarray of peripheral blood mononuclear cells (pbmc) from patients suffering from tuberculosis and m. tuberculosis - infected healthy individuals who had repeated close contact to tuberculosis patients (such as a nurse, physician, or family member) and were tuberculin skin test positive showed that the concentrations of effector molecules, -defensins 1, 3, and 4, were upregulated in the diseased patients . Furthermore, mice infected with 1.5 10 cfu of m. tuberculosis h37rv and treated with different doses of hnp-1 injected subcutaneously showed significantly improved clearance of bacilli from the lungs, liver, and spleen . Hnp-1 at 5 g / ml killed m. avium - intracellulare in vitro at the optimal ph for bactericidal activity (> 5). The minimal inhibitory concentration (mic) of hnp-1 against m. tuberculosis in vitro in one study was 2.5 g / ml, much lower than that reported in a second study (25 g / ml). Hnp was the first defensin found to be effective against nontuberculous mycobacteria, including m. avium - intracellulare . Hnp-2 and hnp-3 were as effective in killing m. avium - intracellulare in vitro as hnp-1 . For hnp-5, the linear peptide derived from its n - terminal fatty acylation can enhance activity against m. tuberculosis almost comparable to the native peptide . Bovine and rabbit defensins have similar or more potent antimycobacterial activity than hnps, especially against m. tuberculosis clinical isolates in vitro [72, 73]. In contrast, another study showed that hnp-13 are not necessarily more effective in killing m. tuberculosis even at a much higher concentration, which may be the result of differing levels of resistance in the individual strains (m. tuberculosis erdman) used . Defensins may play a much more significant role in immunity against mycobacteria than what was previously thought . One can therefore speculate that defensin production, especially by epithelial cells and neutrophils, is likely to be more important early in infection before the establishment of the granuloma . Based on its antimicrobial activity and function in host immunity, neutrophil - macrophage cooperation against m. tuberculosis first, hnps clearly show antimicrobial activity against m. tuberculosis in vitro by increasing the permeability of the mycobacterial cell envelope . Second, in addition to direct antimicrobial activity, hnps secreted by neutrophils recruited into the early lesion are clearly able to act as chemotactic factors, attracting immune cells including macrophages, t - lymphocytes, mast cells, and immature memory t - cells . Third, hnps released by neutrophils recruited in the early lesion can also modulate cytokine production to influence the inflammatory response since tnf- secreted by macrophages may stimulate neutrophil mycobactericidal activity, which might be mediated simultaneously by defensins . In vitro, the capacity of macrophages to control m. tuberculosis growth is improved by transfecting human monocyte - derived macrophages with the hbd-2 gene compared with non - hbd-2-transfected cells ., hbd-1 also plays a role in immunity against m. tuberculosis by permeabilization of both the mycobacterial cell wall and the cell membrane . M. tuberculosis infection of endothelial cells in vitro also results in hbd-1 overexpression and profound cytoskeletal rearrangement . One study found that infection of human limbocorneal fibroblasts with m. tuberculosis, m. abscessus, and m. smegmatis results in overexpression of hbd-13 . Recent studies have emphasized the role of hbd-4, which can be triggered through the il-1 and vitamin d receptor (vdr) pathways in the innate immune defense against m. tuberculosis survival in infected macrophages . One study showed that intratracheal administration of l - isoleucine into mice infected with the antibiotic - sensitive strain h37rv or a multidrug - resistant clinical isolate can significantly upregulate -defensins 3 and 4 and decrease bacillary loads by inducing their gene expression, demonstrating that it may be possible to use defensins for treating infection by modulating their gene expression . Interestingly, more highly virulent m. bovis strains induce lower levels of murine -defensin 4 (mbd4) expression than strains of lower virulence during many time points of early infection, indicating the ability to suppress induction early in infection in vivo . In experimental tuberculosis, expression of mbd3 and mbd4 by airway epithelial cells in the early stages of infection correlated with temporary control of mycobacterial growth . Similarly, high and stable production mbd4 during latent infection is associated with long - term control of mycobacterial proliferation . The activity of defensins against mycobacteria coupled with their induction by infection suggests that the introduction of defensins either prophylactically or early in infection may affect the course of the disease to the benefit of the host . There thus exists the potential for the use of defensins as new prophylactic / therapeutic agents against mycobacterial infections . The effective in vitro activity of a number of defensins against mycobacteria combined with their beneficial chemotactic effects suggests that therapeutic or prophylactic administration of defensins or their induction in the body might lead to improvement in the course of infection and host health . To date, there are relatively few reports on the effects of defensin administration against m. tuberculosis, but most of these show beneficial effects . Mice transfected with the -defensin 2 gene showed higher survival and lower bacterial burden after challenge . Hnp-1 injected postinfection showed significant time- and dose - dependent clearance of bacilli from lungs, livers, and spleens in mice experimentally infected with m. tuberculosis h37rv . The hnp-1 administered to mice in that study was significantly less (1 and 5 g per mouse) than the concentration required (50 g / ml) for antimycobacterial activity in vitro . The higher in vivo potency of hnps is likely due to their immune enhancing effects, such as chemotaxis of t - cells and monocytes . Human defensins are reported to show synergistic activity with antituberculosis drugs, which suggests that they may be a promising adjunct to antituberculosis chemotherapy . A number of studies have explored the combined effect of defensins and antituberculosis drugs against intracellular mycobacteria . In vitro studies suggest that hbd-2 is involved in reducing m. tuberculosis growth, and the combination of hnp-1 with antituberculosis drugs (i.e., isoniazid and rifampicin) resulted in a significant reduction (p <0.001) in mycobacterial load . Hbd-1 has a lower activity against m. tuberculosis, and its combination with isoniazid significantly reduced m. tuberculosis growth in comparison with the peptides or isoniazid alone by permeabilization of both the mycobacterial cell wall and the cell membrane . Moreover, a protective role for -defensin against mycobacterial infection has been reported in human eosinophils . -defensin released by eosinophils upon stimulation with lipomannan from m. bovis bcg, when used with eosinophil cationic protein, showed a synergistic inhibitory effect on mycobacterial growth inhibition . Since the peculiar mycobacterial cell envelope is considered to contribute to the resistance to conventional antimycobacterial drugs, the combination of hnps and antituberculosis drugs against m. tuberculosis h37rv not only results in increased permeability of both the mycobacterial cell wall and the cell membrane but also increases the access to intracellular targets for antituberculosis drugs . Therefore, antimicrobial peptides are potential adjuncts to chemotherapy together with conventional drugs against tuberculosis . Antimicrobial peptides can be more potent in vivo because of their immune enhancing effects by acting as a chemotactic factor and regulatory factor, interacting with immune cells like t - cells and monocytes and modulating the production of cytokines and inflammation . Studies have shown that m. bovis bcg - induced hbd-2 mrna expression in human epithelial cells can influence protection against m. tuberculosis challenge, and m. bovis bcg cell wall components (1830 kda) can stimulate human pulmonary epithelial cells to express defensins . It also shows that prime - boost bcg vaccination with -defensin 2 dna vaccines can enhance the activity against m. tuberculosis . It is known that protection conferred by bcg against tuberculosis is variable and can maintain long - term immunity, and defensins could be important as a component part of this protection against human tuberculosis . Although -defensins have antimicrobial activity against diverse pathogens [35, 93], especially viruses, there is, as yet, no evidence that -defensins are involved in defense against mycobacterial infection . Rivas - santiago et al . Found that vitamin d and l - isoleucine can induce the production of defensins by modulating their gene expression [82, 95]. Therefore, future studies should focus on the mechanism by which defensin gene expression is modulated . On the other hand, although a large number of studies have discovered the antimycobacterial activity of defensins in vitro, there are fewer studies in vivo, and further study should include the activity of defensins against tuberculosis in vivo . Although defensins have been examined for their clinical treatment of infections with no success, defensins have a huge clinical potential, and more research into their application is needed . Defensins are a family of antimicrobial peptides that are abundant amid an array of oxygen - independent antimicrobial proteins and peptides in neutrophil granules and secreted by epithelial cells . The advantages and disadvantages of the various forms of defensin therapy / prophylaxis against mycobacterial infection of man and animals outlined above indicate that this could be an effective new approach to treatment and prevention of these chronic infections, which are becoming increasingly intractable to chemotherapy . Administration of defensins may have direct effects on the pathogens, stimulate innate and adaptive immunity, or be used synergistically with currently used or new chemotherapeutic agents . The experimental work with mycobacterial infections combined with their wide spectrum of activity suggests that bacterial infections other than those caused by mycobacteria may also be amenable to this approach.
The opioid receptors (,,, and the opioid - like receptor orl-1) belong to the super family of g - protein coupled receptors (gpcrs) that possess seven helical trans - membrane spanning domains in their architecture . The majority of research efforts focused upon this group of proteins has been directed toward the receptor because it mediates the analgesic actions of opiates such as morphine (chart 1). Over the years, however, it has become increasingly clear that the entire family of opioid proteins are actively involved in a host of important physiological processes . Studies with selective opioid receptor antagonists have shown that this system is intimately involved in brain processes that relate to stress, fear, and anxiety as well as reward - seeking behavior . Studies have shown that (3r)-1,2,3,4-tetrahydro-7-hydroxy - n-[(1s)-1-[[(3r,4r)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl]-3-isoquinolinecarboxamide (jdtic) and nor - binaltorphimine (nor - bni), another opioid selective antagonist, dose - dependently reduce fear and stress - induced responses in multiple behavioral paradigms with rodents (immobility in the forced - swim assay, reduction of exploratory behavior in the elevated plus maze, and fear - potentiated startle). Further, selective opioid receptor antagonists have been shown to reduce stress - induced reinstatement of cocaine self - administration in rats to block the stress - induced potentiation of cocaine place preference conditioning, to decrease dependence - induced ethanol self - administration, to attenuate the expression of both the physical (somatic signs hyperalgesia) and effective (anxiety - related behavior conditional place aversion) signs of nicotine - induced withdrawal in mice, to diminish deprivation - induced eating in rats, and to prevent prepulse inhibition mediated by u50,488 . These observations regarding the behavioral consequences of receptor blockade in several animal tests suggest that opioid receptor antagonists could be useful for treating anxiety, depression, schizophrenia, addiction, and eating disorders . Compounds 1 (az - mtab),2 (pf-4455242), and 3 (ly2456302) have been reported as newer selective opioid receptor antagonists (chart 1). See also ref (3) for a review of these studies . These newer opioid receptor antagonists show activity in various animal models similar to those reported for norbni and jdtic . In addition, jdtic and compounds 2 and 3 have undergone phase 1 and/or phase 2 studies directed toward various cns disorders . No drugs for the treatment of cocaine and methamphetamine abuse, however, are currently available . Further, nicotine replacement therapy (nrt), bupropion, and varenicline are used to treat nicotine addiction, but no more than 25% of patients respond to these treatments . Naltrexone is used to treat alcoholism but has limited efficacy . A number of antidepressants are on the market, but many patients do not respond to any of them ., we report the synthesis and in vitro efficacy as determined by [s]gtps assay of jdtic analogues 415 (see table 2 for structures). These compounds have the hydroxyl group on the 4-(3-hydroxylphenyl) or 7-hydroxy - tetrahydroisoquinoline parts of jdtic replaced with other functional groups . A comparison of their in vitro efficacy properties to those of jdtic show that several of the analogues were potent and selective opioid receptor antagonists . Preclinical adme studies show that some of the antagonists have better drug - like properties than jdtic . Bis - triflate 17 was prepared by treating 16 with an excess of triflic anhydride at 78 c . Subjection of 17 to palladium - catalyzed transfer hydrogenation in dmf at 80 c afforded intermediate 18 . Reduction of 18 with lithium aluminum hydride in toluene and tetrahydrofuran mixture cleaved the triflamide to give (3r,4r)-3,4-dimethyl-4-phenylpiperidine (19). Reductive amination of 19 with boc - l - valinal, prepared according to the procedure reported by skiles et al ., followed by t - butoxycarbonyl (boc) deprotection with trifluoroacetic acid in dichloromethane afforded 20 . Coupling of 20 with (3r)-2-(tert - butoxycarbonyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (boc-7-hydroxy - d - tic) using n, n, n,n-tetramethyl - o-(1h - benzotriazol-1-yl)uronium hexafluorophosphate (hbtu) in dichloromethane followed by boc deprotection with trifluoroacetic acid in dichloromethane afforded 4 . Scheme 2 shows the synthesis of 8 via intermediate 25, which was also used to synthesize 5, 6, and 7 (scheme 3). Treatment of 22 with n - phenyl(bis - trifluoromethanesulfonimide) afforded the triflate 23 . Amide coupling of 26 with boc-7-hydroxy - d - tic, followed by removal of the boc protecting group with hydrochloric acid in aqueous methanol, afforded the desired final product 8 . The compounds 5, 6, and 7 where the phenolic group of the 4-(3-hydroxyphenyl) group in jdtic has been replaced by a fluoro, chloro, and bromo substituent, respectively, were prepared as described in scheme 3 . Alternatively, sandmeyer halogenation of the diazo intermediate afforded the chloro and bromo intermediates 28 and 29 . Subsequent deprotection of the phthaloyl protected amines present in 27, 28, and 29 using hydrazine in ethanol afforded 30, 31, and 32, respectively . These intermediates were coupled with boc-7-hydroxy - d - tic using hbtu in acetonitrile or 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (edchcl) and a catalytic amount of n - hydroxybenzotriazole (hobt) in dichloromethane followed by treatment with hydrogen chloride to afford 5, 6, and 7 . Coupling of amines 20 and 21(30) with commercially available boc-(3r)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (boc - d - tic), followed by treatment with trifluoroacetic acid in dichloromethane, gave 11 and 9, respectively . Coupling of 21 and 30 with (3r)-2-[tert - butoxy)carbonyl]-7-fluoro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (7-fluoro - boc - d - tic), followed by boc deprotection using aqueous methanolic hydrogen chloride, afforded 10 and 12, respectively . The methyl ester of boc-7-hydroxy - d - tic (33) prepared using trimethylsilyldiazomethane in methanol and toluene was converted to the intermediate aryl triflate with triflic anhydride, which was transformed to the benzonitrile (35) via palladium - catalyzed cyanation . Careful hydrolysis of the methyl ester using lithium hydroxide in aqueous dioxane, followed by addition of hydrogen peroxide to the cooled solution, resulted in a very rapid hydrolysis of the benzonitrile to the benzamide 36 . The appropriate amine (21, 3-{(3r,4r)-1-[(2s)-2-amino-3-methylbutyl]-3,4-dimethylpiperidin-4-yl}benzamide, or 20) could then be coupled with 36 using hbtu or edchcl to afford the intermediate amides which yielded the desired compounds 13, 14, and 15 upon deprotection of the boc group with trifluoroacetic acid in dichloromethane or hydrochloric acid in aqueous methanol . Because [s]gtps binding strongly correlates with animal behavior studies of previously reported antagonists, measures of opioid receptor antagonism and specificity for the compounds in the study were obtained by monitoring the ability of selected test compounds to inhibit stimulation of [s]gtps binding produced by the selective agonists (d - ala, mephe, gly - ol)encephalin (damgo, receptor) cyclo[d - pen, d - pen]encephalin (dpdpe,) and 5,7,8-()-n - methyl - n-[7-(1-pyrrolidinyl)-1-oxaspirodec-8-yl]benzeneacetamide (u69,593,) in cloned human receptors using previously reported methods.ke values were calculated as previously reported . Several in vitro studies were conducted to characterize opioid receptor antagonists 4, 5, 13, and 14 and compared to the results from jdtic and previously reported 37(33) (chart 2), which like compounds 13 and 14 has its phenol groups replaced by a carboxamido group . An in vitro model using mdck - mdr1 cells was used to predict brain penetration . Plasma and s9 stability of each compound was determined using procedures similar to those previously reported . Compounds that interact with the human ether - a - go - go gene herg product (which is a potassium channel) are cardiotoxic . Thus, the affinity of synthesized opioid receptor antagonists toward the herg channel was determined . The interaction of these test compounds with the herg channel was analyzed using a radioligand displacement assay based on a protocol developed by chiu et al . For these studies, [h]astemizole was used as the high - affinity herg radioligand (ki 20 nm). See experimental section for details . Solubility of the compounds was determined using a kinetic 96-well plate assay essentially as described by zhu et al . The parallel artificial membrane permeability assay (pampa) was used to predict oral absorption in a 96-well format as has been described previously and detailed in the experimental section . In the late 1970s, zimmerman and co - workers reported that n - methyl - trans-4-phenylpiperidine 38 (ly83577) (chart 2) was an opioid receptor pure antagonist whose potency was significantly increased by adding a phenolic group to the aromatic ring to give the n - methyl - trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine 39 (ly99335) (chart 2). In a study directed toward determining if the 3-hydroxy group present on the aromatic ring in 40 (ly255582) was required for potent antagonist efficacy, it was found that removal of the 3-hydroxy group led to 40-, 135-, and 39-fold reduction in the kb value at the,, and receptors relative to 40 using [s]gtps assays (table 1). In addition, replacement of the 3-hydroxy group in 40 with 10 other functional groups all led to much reduced in vitro antagonist efficacy relative to 40, suggesting that the phenolic group was essential for the high potency of 40 ., we demonstrate that 4, which has the hydroxyl group of the 4-(3-hydroxyphenyl) group in jdtic replaced by a hydrogen (table 2), is a potent and selective opioid receptor antagonist . Compound 4 has ke values of 8.9, 442, and 0.024 nm at the,, and receptors, respectively, compared to ke values of 25, 74, and 0.02 nm for jdtic . Compound 4 with 370- and 18400-fold selectivity for the receptor relative to the and receptors was more selective than jdtic for the receptor but a little less selective than jdtic for the receptor . Both compounds, however, are highly selective for the receptor relative to both the and receptors . This discovery is in contrast to previously reported structural activity studies of 40 as well as other compounds in this class of compounds . The data represents the mean (se) from at least three independent experiments . Data taken from ref (33). Compound 5, which has a fluoro group in place of the hydrogen present in 4 or the hydroxyl group in jdtic, with ke values of 14.8, 249, and 0.01 nm at the,, and receptors, respectively, and 1480- and 24900-fold selectivity relative to the and receptors, is both a more potent and more selective opioid receptor antagonist than jdtic or 4 . Compounds 6 and 7 have a chloro and bromo group, respectively, in place of the hydroxyl group in jdtic . Compound 6 has a ke = 0.039 nm at the receptor, with 175- and 17600-fold selectivity for the relative to the and receptors and thus has good potency and selectivity . Compound 7, which has the larger bromo group in place of the hydroxyl in jdtic, has a weaker ke value of 0.268 nm at the receptor and only a 25-fold selectivity for the receptor relative to the receptor . Replacement of the hydroxyl group in jdtic with the amino electron donating amino group to give 8 results in a decrease in potency at the receptor (ke = 0.25 nm) and increased potency at the receptor (ke = 10.6 nm). This results in only 42-fold selectivity for the relative to the receptor . Compound 8 has 7600-fold selectivity for the relative to the receptor and thus is more selective than jdtic for the relative to the receptor . In a previous report, we compared the opioid receptor antagonist efficacy of compounds 4145 (chart 2) using the same conditions as that used in this study . The nitro (41), acetylamino (42), methanesulfonylamino (43), and amino (44) analogues were 320-, 70-, 200-, and 10-times less potent as a antagonist than jdtic and were not as selective for the receptor relative to the and receptors as jdtic . The methoxy (45) analogue was only 3-fold less potent than jdtic as a antagonist . Compound 45 was selective for the receptor relative to the and but was not as selective as jdtic . The critical importance of a methoxy or hydroxyl group in the tetrahydroisoquinoline carboxamide (tic) part of jdtic is further shown by the results with 9 and 10, where the tic hydroxyl group in jdtic is replaced by a hydrogen or fluoro substituent, respectively . Compound 9 has ke values of 16, 158, and 4.3 nm at the,, and receptors, respectively, with only 3.7- and 37-fold selectivity for the relative to the and receptors . Thus, 9 is much less potent and selective as a opioid antagonist than jdtic or 4 (compound 9 was previously characterized as the free base but was not evaluated for opioid antagonst efficacy under conditions used in this study). Compound 10 with ke values of 7.7 and 2.20 nm at the and receptors, respectively, is also much less potent and selective as a opioid receptor antagonist . Compound 11 can be viewed as a compound having both hydroxyl groups in jdtic replaced by a hydrogen or by replacement of the tic hydroxyl group in 4 with a hydrogen . Viewing the change either way again shows the importance of the tic hydroxyl group to the high potency and selectivity . Compound 11 with a ke value of 16 nm at the receptor has low potency for this receptor . Compound 12 can be viewed as a compound having both hydroxyl groups in jdtic replaced by fluoro groups or by replacement of the tic hydroxyl group in 5 with a fluoro group . Regardless of how 12 is viewed, its low potency (ke = 2.22 nm compared to 0.02 nm for jdtic) shows the importance of tic hydroxyl to the potency and selectivity of jdtic, 4, and 5 . Similar to 10, 12 also behaved as a weak inverse agonist in the opioid receptor assay . Of all the jdtic analogues synthesized and evaluated herein, these are the only two compounds that are inverse agonists in the receptor assay . One interesting finding from the x - ray crystallographic structure of the human opioid receptor is that the interaction of ligand hydroxyls with the receptor is mediated by intervening structured water molecules (figure 1a, receptor pocket waters indicated by blue green spheres). As illustrated in the two - dimensional kor - jdtic interaction diagram of the 7-hydroxy - d - tic, hydroxyl participates in hydrogen bonds with two structured water molecules which in turn interact with residues lys227 and tyr139 (figure 1b). This binding arrangement suggested that replacing the ligand hydroxyls with a substituent, which could replace the structured water in the x - ray structure, both in location and hydrogen bonding capacity, would result in direct ligand - to - receptor hydrogen bonding interactions . Removing the dependency on water molecules in the receptor pocket might result in enhanced or altered properties . Three of the compounds, 13, 14, and 15, were prepared to test this hypothesis by replacing one or both of the jdtic hydroxyls with carboxamide substituents . Water pair with a carboxamide group was tested by computational docking studies of compound 13 (in which the 7-hydroxyl of 7-hydroxy - d - tic is replaced by a carboxamide). As anticipated, the overall binding pose of compound 13 is identical to that observed for jdtic, with the 13 carboxamide group directly providing a hydrogen - bond interaction with lys227 (figure 2a). The two - dimensional kor-13 interaction diagram of the docking result (figure 2b) illustrates that 13 carboxamide interaction with lys227 may not require an intervening water molecule . (a) three - dimensional view of the hydrogen - bonding interactions between jdtic and the kor (pdb 4djh). (b) two - dimensional diagram of the hydrogen - bond and hydrophobic interactions between the tic moiety of jdtic and the kor (pdb 4djh). The origin of hydrophobic interactions are indicated by the direction of red line segments around the receptor residues and ligand atoms . (a) three - dimensional view of the hydrogen - bonding interactions between compound 13 and the kor (docking calculation). (b) two - dimensional diagram of the hydrogen - bond and hydrophobic interactions between the tetrahydroisoquinoline-7-carboxamide group of compound 13 and the kor (docking calculation). The origin of hydrophobic interactions are indicated by the direction of red line segments around the receptor residues and ligand atoms . The his291 nitrogen - to - carboxamide nitrogen distance (d1) is 3.89 and the lys227 carbonyl oxygen - to - carboxamide nitrogen distance (d2) is 2.83 . In this study, we found that 13 had a ke = 0.02 nm and thus was as potent a opioid receptor antagonist as jdtic (table 2). Compound 14, which has both phenolic groups in jdtic replaced by a carboxamide group, has a ke = 0.11 nm at the opioid receptor . All three compounds are highly selective for the relative to the and receptors . Compound 15 can be viewed as an analogue of 4, where the hydroxyl group in the tic portion of 7 has been replaced by a carboxamide group . This compound with a ke = 0.041 nm at the receptor and 163- and 2700-fold selectivity for the receptor relative to the and receptors, respectively, is slightly less potent and selective than compound 4 . In previous studies, we reported that 37, which has a carboxamide group replacing the hydroxyl of the 4-(3-hydroxyphenyl) group, has a ke = 0.12 nm at the receptor, which was only 6 times less potent than jdtic as a opioid receptor (kor) antagonist (table 2). Calculated physiochemical properties such as topological polar surface area (tpsa), lipophilicity (clogp), and derived values such as logbb are useful indicators of a compound s potential to penetrate the brain . These molecular descriptors were calculated for jdtic, previously reported 37, as well as 4, 5, 13, and 14 (table 3). In general, cns drugs have clogp in the range 24, tpsa less than 76, and logbb greater than 1 . The lead compound, jdtic, which proceeded through phase 1 clinical studies, has a tpsa = 84.83, which is larger than 76 . Compounds 13, 14, and 37, with tpsa values of 107.69, 130.55, and 107.69, respectively, are also above the 76 . Compounds 4 and 5 both have tpsa values of 64.6, which is less than 76 . Jdtic and all of the analogues except 5 had clogp values of less than 4 . Jdtic, 4, and 5 have logbb values of 0.57, 0.23, and 0.19 and thus are greater than 1, predicting good brain penetration . Compounds 13, 14, and 37 with logbb values of 0.98, 1.39, and 1.02, are predicted to have poorer brain penetration . Compounds that interact with the human ether - a - go - go gene (herg) product, which is a potassium channel, can produce qt prolongation and cardiotoxic effects . Compounds 13, 14, and 37 have ki values of> 10 m (table 4). Compounds 4 and 5 have ki values of 7.05 and 6.25 m, similar to the 8.82 m value for jdtic (table 4). Compounds that have> 5% permeability in the mdck assay,> 50% stability in the plasma and s9 stability assay,> 25% transported in the pampa assay, and> 20 m solubility are considered desirable . All of the jdtic analogues except previously reported 37 have> 5% permeability in the mdck assay . Compounds 4, 13, and 14, with 27, 17, and 14%, were more permeable than jdtic, which had 11% permeability (table 4). All of the compounds showed> 20 m solubility at ph 3, and all compounds were more soluble than jdtic, which was considered to be highly soluble . Compounds 13, 14, and 37 all also had> 20% solubility at ph 7.4 . Compounds 4 and 5 had values of 11 and 10 m, which are similar to the 11 m for jdtic . Jdtic and the synthesized analogues showed good stability in both the plasma and s9 stability assays . Jdtic, 4, 13, and 14 had> 25% transported at both ph 5.5 and 7.4 in the pampa assay . On the basis of the mdck, solubility, plasma and s9 stability, and pampa results combined with the calculated tpsa, clogp, and logbb data, 4 appears to have the best overall profile . However, the ki = 7.05 m in the herg could be of concern . Compounds 13 and 14 show very favorable mdck, solubility, plasma and s9 stability, and pampa results as well as ki values of> 10 m for binding in the herg assay . However, the calculated tpsa and logbb suggest that brain penetration could be a concern . Previously reported 37 was evaluated for its ability to block agonist u50,488-induced diuresis at 330 mg / kg ig and 130 mg / kg ip in rats . Compound 37 blocked the u50,488-induced diuresis at 24 h and 8 days at 1, 10, and 30 mg / kg, 15 days at 10 and 30 mg / kg, and 22 and 29 days at 30 mg / kg following ip administration . Compound 37 was ineffective in blocking the u50,488-induced diuresis when given ig . Because opioid receptor agonist - induced diuresis is mediated by the central nervous system, the high potency of 37 in this assay after ip administration suggest that the high tpsa valve (107.69) and logbb (1.02) are misleading for this jdtic analogue having an aromatic carboxamido substituent . Similarly, the high tpsa and logbb values for 13 and 14, both of which have aromatic carboxamido substituents, could also be misleading . The lack of efficacy after oral (ig) administration of 37 could be due to its poor pampa values (1.2 and 3.2% at ph 7.4 and 5.5, respectively) and its poor mdck value (2%). In summary, these studies provide the unexpected finding that replacement of the 3-hydroxyl substituent of the 4-(3-hydroxphenol) group of jdtic with either a hydrogen, fluoro, or chloro group leads to opioid receptor antagonists that are as highly potent and selective as jdtic . This finding is in contrast to what would have been predicted based on structure activity relationship studies of other n - substituted 3,4-dimethyl-4-(3-hydroxyphenyl)piperidines such as 40 (ly255582), as well as much of the sar studies reported for opioid ligands in general . The high opioid receptor potency and selectivity relative to the and opioid receptors of 4, 5, and 14 combined with their favorable herg, mdck, pampa, solubility, and plasma and s9 stability in vitro preclinical studies and calculated tpsa, clogp, and logbb values suggest that the compounds should be considered for further development as potential drugs for treating depression, anxiety, schizophrenia, and addiction (cocaine, nicotine, methamphetamine, alcohol, and eating disorders). Melting points were determined using a mel - temp ii capillary melting point apparatus and are uncorrected . Nuclear magnetic resonance (h nmr and c nmr) spectra were obtained on a varian avance dpx-500 mhz nmr spectrometer or a bruker unity inova 300 mhz nmr spectrometer . Chemical shifts are reported in parts per million (ppm) with reference to internal solvent . Mass spectra (ms) were conducted on a perkinelmer sciex ap1 150 ex mass spectrometer equipped with esi (turbospray) source . Atlanta, ga . The purity of the compounds (> 95%) was established by elemental analysis . Optical rotations were measured on an autopol iii polarimeter, purchased from rudolf research . Analytical thin - layer chromatography (tlc) was carried out using emd silica gel 60 f254 tlc plates . Flash column chromatography was done on a combiflash companion system using isco prepacked silica gel columns or using em science silica gel 60a (230400 mesh). Unless otherwise stated, reagent - grade chemicals were obtained from commercial sources and were used without further purification . All moisture- and air - sensitive reactions and reagent transfers were carried out under dry nitrogen . The amine 20 (137 mg, 0.50 mmol), boc-7-hydroxy - d - tic (161 mg, 0.55 mmol), hbtu (208 mg, 0.55 mmol), and net3 (280 l, 2.2 mmol) were stirred in ch2cl2 (11 ml) for 12 h. the concentrated residue was subjected to chromatography on silica gel using a step gradient up to 50% cma80 in ch2cl2 to afford the boc - protected intermediate . This boc - protected compound was stirred in tfa / ch2cl2 (1:1, 10 ml) for 12 h, concentrated, and the residue subjected to chromatography on silica gel (12 g) using a gradient up to 50% cma80 in ch2cl2 as the eluent to afford the free base of 4 . H nmr (300 mhz, cdcl3) 7.087.34 (m, 6h), 6.836.95 (m, 1h), 6.586.68 (m, 1h), 6.436.54 (m, 1h), 3.954.12 (m, 1h), 3.82 (s, 2h), 3.333.49 (m, 1h), 2.953.15 (m, 1h), 2.442.91 (m, 5h), 2.202.43 (m, 2h), 1.842.03 (m, 2h), 1.60 (d, j = 13.0 hz, 1h), 1.29 (s, 3h), 0.820.98 (m, 6h), 0.640.79 (m, 3h). C nmr (75 mhz, cdcl3) 173.3, 154.9, 136.6, 130.2, 128.1, 125.7, 125.7, 125.4, 125.2, 114.2, 112.3, 59.6, 57.1, 55.5, 51.2, 50.6, 47.9, 38.8, 38.4, 30.9, 30.7, 30.4, 27.7, 19.1, 17.8, 16.3 . Ms (esi) m / z 450.7 (m + h). The free base was converted to 67.7 mg (24%) of the dihydrochloride salt (over two steps) as a white powder: mp 195199 c, []d + 101 (c 0.17, ch3oh). (c28h41cl2n3o22h2o) c, h, n. the amine 30 (146 mg, 0.50 mmol) was combined with boc-7-hydroxy - d - tic (150 mg, 0.54 mmol) and hbtu (200 mg, 0.53 mmol) and dissolved in ch3cn (10 ml) before net3 (0.2 ml, 1.4 mmol) was added . The concentrated residue was subjected to chromatography on silica gel using a gradient of etoac in hexanes to afford an oil (225 mg) which was dissolved in ch3oh (3 ml) and 6 n hcl (3 ml) and stirred 12 h. the concentrated residue was dissolved in dilute nh4oh and extracted with ch2cl2 . The residue was subjected to chromatography on silica gel using etoac then a gradient of cma80 in ch2cl2 as the eluent to afford the free base of 5 . H nmr (cdcl3) 7.177.26 (m, 1h), 7.11 (d, j = 9.0 hz, 1h), 7.01 (d, j = 8.1 hz, 1h), 6.786.97 (m, 3h), 6.65 (d, j = 8.3 hz, 1h), 6.51 (s, 1h), 4.03 (dd, j = 4.9, 9.8 hz, 1h), 3.87 (s, 2h), 3.45 (dd, j = 4.7,10.9 hz, 1h), 3.07 (dd, j = 4.5, 16.0 hz, 1h), 2.542.80 (m, 3h), 2.382.52 (m, 3h), 2.142.37 (m, 2h), 1.842.01 (m, 2h), 1.54 (d, j = 12.6 hz, 1h), 1.221.33 (m, 4h), 0.92 (dd, j = 7.0, 8.9 hz, 6h), 0.68 (d, j = 6.8 hz, 3h). C nmr (75 mhz, cdcl3) 173.2, 163.0 (d, j = 244 hz), 155.1, 153.1 (d, j = 6.5 hz), 136.7, 130.1, 129.4 (d, j = 8.4 hz), 125.2, 121.3 (d, j = 2.3 hz), 114.2, 112.8 (d, j = 21.8 hz), 112.3, 112.1 (d, j = 19.5 hz), 59.5, 57.1, 55.4, 51.1, 50.8, 47.9, 38.8, 30.8, 30.5, 30.4, 27.5, 19.2, 17.7, 16.2 . Ms (esi) m / z 468.1 (m + h). The free base was converted to the dihydrochloride salt (100 mg, 34% over two steps) as a white powder . F nmr (282 mhz, dmso - d6) 112.97; mp 219223 c (fusion); []d = + 174 (c 0.4, ch3oh). (c28h40cl2fn3o23h2o) c, h, n. to a solution of 31 (56 mg, 0.18 mmol) and boc-7-hydroxy - d - tic (59 mg, 0.20 mmol) in ch2cl2 (10 ml) was added edchcl (77 mg, 0.40 mmol), hobt (3 mg, 0.02 mmol), and net3 (115 l, 0.82 mmol). The reaction mixture was stirred at ambient temperature for 12 h then was diluted with ch2cl2 (20 ml) and washed with aq nahco3 (10 ml). The residue was subjected to chromatography on silica gel using a gradient up to 50% cma80 in chcl3 to afford the 76 mg (72%) of the boc - protected intermediate as a white solid . The intermediate was dissolved in acetonitrile (5 ml) and treated with hcl in dioxane (4.0 m, 0.33 ml). The resulting residue was subjected to chromatography on silica using a gradient up to 50% cma80 in chcl3 as the eluent to afford 35 mg (48%) of the free base 6 as white solid . H nmr (cd3od) 7.137.45 (m, 4h), 7.01 (d, j = 8.1 hz, 1h), 6.68 (d, j = 7.5 hz, 1h), 6.58 (br s, 1h), 4.93 (br s, 2h), 4.044.43 (m, 3h), 3.96 (br s, 1h), 3.23 (br s, 1h), 3.073.19 (m, 3h), 3.02 (br s, 2h), 2.53 (br s, 1h), 2.30 (br s, 1h), 1.90 (br s, 1h), 1.661.86 (m, 1h), 1.41 (s, 3h), 0.99 (br s, 6h), 0.640.88 (m, 3h). C nmr (cd3od) 175.3, 162.1, 155.3, 139.7, 135.1, 133.6, 131.4, 130.6, 128.9, 125.7, 120.9, 117.6, 66.3, 61.4, 58.8, 56.9, 55.0, 49.4, 42.8, 42.3, 36.0, 33.8, 32.4, 30.8, 23.7, 22.2, 19.6 . Ms (esi) the product was converted to the dihydrochloride salt: []d = + 92.0 (c 0.52, meoh). (c28h40cl3n3o22.5h2o) c, h, n). To a solution of 32 (94 mg, 0.26 mmol) and boc-7-hydroxy - d - tic (89 mg, 0.30 mmol) in ch2cl2 (10 ml) was added edchcl (116 mg, 0.60 mmol), hobt (5 mg, 0.03 mmol), and triethylamine (173 l, 1.24 mmol). The reaction mixture was stirred at ambient temperature for 12 h. the reaction mixture was diluted with ch2cl2 (20 ml) and washed with aq nahco3 (10 ml). The organic layer was dried (na2so4) and concentrated to a residue which was subjected to chromatography on silica gel using a gradient up to 50% cma80 in chcl3 as the eluent to afford 79 mg (46%) of the boc - protected intermediate as a white solid . H nmr (cdcl3) 7.137.27 (m, 2h), 7.09 (d, j = 4.90 hz, 2h), 6.91 (d, j = 8.3 hz, 1h), 6.506.75 (m, 1h), 6.45 (br s, 1h), 4.384.54 (m, 1h), 4.34 (br s, 1h), 3.78 (br s, 1h), 3.11 (dd, j = 3.0, 15.1 hz, 1h), 2.90 (dd, j = 5.8, 15.3 hz, 1h), 2.50 (br s, 1h), 2.272.42 (m, 1h), 2.24 (br s, 1h), 1.932.14 (m, 3h), 1.591.93 (m, 2h), 1.43 (s, 9h), 1.35 (br s, 1h), 1.14 (s, 3h), 0.78 (d, j = 6.6 hz, 3h), 0.69 (d, j = 6.8 hz, 3h), 0.52 (d, j = 6.8 hz, 3h). C nmr (cdcl3) 171.3, 155.7, 152.9, 134.0, 129.7, 129.3, 128.8, 128.4, 124.4, 122.5, 114.8, 112.9, 81.6, 59.9, 56.1, 51.3, 50.0, 44.8, 38.8, 38.7, 30.5, 29.8, 28.4, 27.4, 19.2, 16.9, 16.0 . Ms (esi) m / z 628.6 (m + h). The intermediate was dissolved in acetonitrile (5 ml) and treated with hcl in dioxane (4.0 m, 0.3 ml) and stirred for 12 h. concentration of the reaction mixture afforded a white solid which was chromatographed on silica using a gradient up to 50% cma80 in chcl3 to afford 56 mg (88%) of 7 free base as white solid . H nmr (cdcl3) 7.147.27 (m, 2h), 6.957.14 (m, 2h), 6.84 (d, j = 8.3 hz, 1h), 6.486.66 (m, 1h), 6.43 (d, j = 2.1 hz, 1h), 3.944.23 (m, 1h), 3.80 (br s, 2h), 3.38 (dd, j = 4.7, 10.7 hz, 1h), 3.00 (dd, j = 4.2, 16.3 hz, 1h), 2.70 (br s, 1h), 2.57 (dd, j = 11.0, 14.9 hz, 2h), 2.43 (br s, 2h), 2.27 (d, j = 8.10 hz, 1h), 2.17 (br s, 1h), 1.98 (s, 1h), 1.87 (br s, 2h), 1.49 (d, j = 12.2 hz, 1h), 0.971.28 (m, 3h), 0.700.91 (m, 6h), 0.62 (d, j = 6.8 hz, 3h). C nmr (cdcl3) 173.1, 154.5, 152.5, 136.8, 130.2, 129.7, 128.9, 128.5, 125.6, 124.4, 122.6, 114.0, 112.2, 59.4, 57.0, 55.2, 51.1, 50.6, 47.9, 38.7, 38.6, 30.6, 30.3, 27.5, 19.2, 17.8, 16.2 . The free base was converted to the dihydrochloride salt: []d = + 98.0 (c 0.61, meoh). (c28h40brcl2n3o20.5h2o) c, h, n. to a solution 26 (125 mg, 0.43 mmol) in ch2cl2 (10 ml) was added boc-7-hydroxy - d - tic (125 mg, 0.43 mmol), hobt (10 mg, 0.1 mmol), and edchcl (191 mg, 1.0 mmol), followed by the addition of diisopropylethylamine (0.15 ml, 0.86 mmol). The resulting solution was stirred at room temperature for 12 h then washed with saturated aqueous nahco3 (5 ml). The combined organic layers were washed with brine (5 ml), dried (na2so4), and concentrated . The resulting residue was purified by chromatography on silica gel using a gradient up to 50% cma80 in ch2cl2 as the eluent . The product containing fractions were combined and concentrated to afford 174 mg (72%) of the boc - protected intermediate . The intermediate was then dissolved in ch3oh (10 ml) to which aq hcl (6 n, 10 ml) was added . The resulting residue was subjected to chromatography on silica gel using a gradient up to 50% cma80 in ch2cl2 as the eluent to afford 8 free base . H nmr (300 mhz, cdcl3) 6.997.18 (m, 2h), 6.85 (d, j = 8.3 hz, 1h), 6.416.72 (m, 5h), 4.02 (dt, j = 4.6, 9.1 hz, 1h), 3.82 (s, 2h), 3.383.48 (m, 2h), 3.02 (dd, j = 4.7, 16.2 hz, 1h), 2.512.79 (m, 3h), 2.362.51 (m, 3h), 2.102.35 (m, 2h), 1.792.00 (m, 2h), 1.50 (d, j = 12.6 hz, 1h), 1.161.31 (m, 3h), 0.830.98 (m, 6h), 0.66 (d, j = 6.8 hz, 3h). C nmr (75 mhz, cdcl3) 173.1, 154.9, 151.3, 145.8, 136.4, 130.0, 128.8, 124.9, 116.4, 114.2, 112.9, 112.4, 112.2, 59.4, 56.8, 55.3, 51.2, 50.7, 47.6, 38.7, 38.2, 30.7, 30.5, 30.1, 27.3, 19.0, 17.7, 16.2 . The free base was converted to the trihydrochloride salt, affording 132.4 mg (50% over two steps) of a white powder . Ms (esi) m / z 465.5 (m + h), mp 241243 c (fusion), []d + 98.7 (c 0.38, ch3oh). (c28h43cl3n4o22.5h2o) c, h, n. the amine 21 (145 mg, 0.50 mmol), boc - d - tic (152 mg, 0.55 mmol), hbtu (208 mg, 0.55 mmol), and net3 (280 l, 2.2 mmol) were stirred in ch2cl2 (11 ml) for 12 h. the concentrated residue was subjected to chromatography on silica gel using a step gradient up to 50% cma80 in ch2cl2 as the eluent to afford the boc - protected intermediate . This boc - protected compound was stirred in tfa / ch2cl2 (1:1, 10 ml) for 12 h, concentrated, and the residue subjected to chromatography on silica gel (12 g) using a gradient up to 50% cma80 in ch2cl2 as the eluent to afford the free base of 9 . H nmr (300 mhz, cdcl3) 6.967.21 (m, 5h), 6.736.82 (m, 2h), 6.596.67 (m, 1h), 4.07 (dt, j = 4.9, 9.3 hz, 1h), 4.00 (s, 2h), 3.56 (dd, j = 5.1, 10.7 hz, 1h), 3.20 (dd, j = 5.0, 16.7 hz, 1h), 2.702.83 (m, 2h), 2.64 (d, j = 10.4 hz, 1h), 2.292.49 (m, 4h), 2.20 (dt, j = 4.3, 12.4 hz, 1h), 1.802.01 (m, 2h), 1.54 (d, j = 13.0 hz, 1h), 1.231.30 (m, 3h), 0.93 (dd, j = 6.8, 9.0 hz, 6h), 0.67 (d, j = 6.8 hz, 3h). C nmr (75 mhz, cdcl3) 172.9, 155.9, 152.1, 135.8, 134.4, 129.3, 129.1, 126.5, 126.1, 125.5, 117.7, 113.1, 112.4, 59.6, 56.7, 55.1, 51.1, 50.6, 47.8, 38.9, 38.5, 31.1, 30.6, 27.5, 19.2, 17.8, 16.2 . Ms (esi) m / z 450.5 (m + h). The free base was converted to the dihydrochloride salt (50.3 mg, 18% over two steps) as a white powder: mp 197200 c (fusion), []d = + 108 (c 0.10, ch3oh). (c28h41cl2n3o22h2o) c, h, n. amine 21 (145 mg, 0.50 mmol) and acid 7-fluoro - boc - d - tic (162 mg, 0.55 mmol) were combined in ch2cl2 (10 ml) and treated with edchcl (191 mg, 1.0 mmol) then net3 (0.35 ml, 2.5 mmol). After 12 h, the concentrated residue was subjected to chromatography on silica gel using a gradient up to 60% cma80 in ch2cl2 as the eluent . The product containing fractions were concentrated then treated with meoh (5 ml) and aq hcl (6 n, 5 ml). After 1 h, the concentrated residue was subjected to chromatography on silica gel using a gradient up to 75% cma80 in ch2cl2 as the eluent to afford 10 free base . H nmr (300 mhz, cdcl3) 7.067.22 (m, 2h), 7.01 (dd, j = 5.8, 8.3 hz, 1h), 6.616.86 (m, 5h), 4.09 (tt, j = 4.7, 9.4 hz, 1h), 3.95 (s, 2h), 3.52 (dd, j = 4.9, 10.6 hz, 1h), 3.14 (dd, j = 4.8, 16.5 hz, 1h), 2.612.82 (m, 3h), 2.272.55 (m, 4h), 2.112.27 (m, 1h), 1.801.98 (m, 2h), 1.52 (d, j = 12.8 hz, 1h), 1.25 (s, 3h), 0.92 (t, j = 7.7 hz, 6h), 0.66 (d, j = 6.8 hz, 3h). C nmr (75 mhz, cdcl3) 172.9, 161.2 (d, j = 245 hz), 156.4, 151.9, 137.4 (d, j = 6.5 hz), 130.7 (d, j = 7.8 hz), 129.7 (d, j = 2.9 hz), 129.1, 117.3, 113.6 (d, j = 21.2 hz), 113.1, 112.6, 112.1 (d, j = 21.1 hz), 59.6, 56.7, 55.1, 51.3, 50.6, 47.6, 38.8, 38.4, 30.8, 30.7, 30.3, 27.5, 19.2, 17.8, 16.3 . The free base was converted to the dihydrochloride salt (90.7 mg, 15% over two steps) as a white powder: mp 202206 c (fusion); []d = + 93 (c 0.1, ch3oh). (c28h40cl2fn3o22.5h2o) c, h, n. the amine 20 (137 mg, 0.50 mmol), boc - d - tic (152 mg, 0.55 mmol), hbtu (208 mg, 0.55 mmol), and net3 (280 l, 2.2 mmol) were stirred in ch2cl2 (11 ml) for 12 h. the concentrated residue was subjected to chromatography on silica gel using a step gradient up to 50% cma80 in ch2cl2 as the eluent to afford the boc - protected intermediate . This boc - protected compound was stirred in tfa / ch2cl2 (1:1, 10 ml) for 12 h, concentrated, and the residue subjected to chromatography on silica gel (12 g) using a gradient up to 50% cma80 in ch2cl2 as the eluent to afford the free base of 11 . H nmr (300 mhz, cdcl3) 7.207.34 (m, 4h), 7.067.19 (m, 3h), 6.967.05 (m, 1h), 4.034.10 (m, 1h), 4.00 (s, 2h), 3.53 (dd, j = 4.9, 10.9 hz, 1h), 3.133.25 (m, 1h), 2.88 (d, j = 15.1 hz, 1h), 2.672.84 (m, 3h), 2.58 (d, j = 10.4 hz, 3h), 2.262.48 (m, 3h), 2.002.09 (m, 1h), 1.852.01 (m, 1h), 1.561.69 (m, 1h), 1.31 (s, 3h), 0.880.99 (m, 6h), 0.70 (d, j = 7.0 hz, 2h). C nmr (75 mhz, cdcl3) 172.9, 135.9, 134.4, 129.3, 128.2, 126.4, 126.1, 125.6, 125.5, 59.2, 56.8, 55.1, 51.2, 50.3 . 47.9, 38.6, 38.3, 31.2, 30.7, 27.5, 19.2, 17.9, 16.1 . The free base was converted to the dihydrochloride salt (21.9 mg, 8% over two steps) as a white powder: mp 162165 c (fusion), []d = + 96 (c 0.10, ch3oh). (c28h41cl2n3o2h2o) c, h, n. amine 30 (24.2 mg, 0.083 mmol) and acid 7-fluoro - boc - d - tic (55.7 mg, 0.19 mmol) were combined in ch2cl2 (8 ml) and treated with edchcl (40 mg, 0.2 mmol) then net3 (0.10 ml, 0.72 mmol). After 12 h, the concentrated residue was subjected to chromatography on silica gel using a gradient up to 50% cma80 in ch2cl2 . The product containing fractions were concentrated then treated with meoh (5 ml) and aq hcl (5 ml). After 1 h, the concentrated residue was subjected to chromatography on silica gel using a gradient up to 50% etoac in hexanes with 1% nh3 (prepared by adding 1% concd nh4oh by volume as the eluent and drying over na2so4) as the eluent to afford 12 free base . H nmr (300 mhz, cdcl3) 7.197.29 (m, 1h), 6.987.14 (m, 3h), 6.93 (td, j = 2.1, 11.3 hz, 1h), 6.84 (tt, j = 2.8, 8.3 hz, 2h), 6.73 (dd, j = 2.6, 9.0 hz, 1h), 3.944.10 (m, 3h), 3.51 (dd, j = 4.9, 10.6 hz, 1h), 3.15 (dd, j = 5.0, 16.5 hz, 1h), 2.582.85 (m, 3h), 2.062.54 (m, 5h), 1.852.02 (m, 2h), 1.56 (dd, j = 1.2, 12.9 hz, 1h), 1.28 (s, 3h), 0.880.99 (m, 6h), 0.66 (d, j = 7.0 hz, 3h). C nmr (75 mhz, cdcl3) 172.5, 163.2 (d, j = 244.4 hz), 161.3 (d, j = 244.7 hz), 153.3 (d, j = 6.4 hz), 137.7 (d, j = 6.5 hz), 130.8 (d, j = 7.8 hz), 130.0 (d, j = 2.9 hz), 129.6 (d, j = 8.2 hz), 121.3 (d, j = 2.6 hz), 113.7 (d, j = 21.2), 112.8 (d, j = 21.4 hz), 112.3 (d, j = 21.1 hz), 112.2 (d, j = 21.1 hz), 59.5, 56.7, 55.2, 51.2, 50.6, 47.7, 38.7, 38.7, 30.6, 30.5, 30.4, 27.4, 19.2, 17.8, 16.2 . Ms (esi) m / z 470.9 (m + h). The free base was converted to the dihydrochloride salt (21.9 mg, 46% over two steps) as a white powder: mp 152156 c (fusion), []d = + 115 (c 0.10, ch3oh). (c28h39cl2f2n3o2h2o) c, h, n. the amine 21 (153 mg, 0.53 mmol) was added to a solution of the acid 36 (170 mg, 0.53 mmol), hbtu (209 mg, 0.55 mmol), and net3 (0.24 ml, 1.7 mmol) in ch3cn (40 ml). After 12 h, the residue obtained on concentration was subjected to chromatography on silica gel using a gradient up to 70% cma80 in ch2cl2 as the eluent . The product containing fractions were dissolved in ch2cl2 (5 ml) and tfa (5 ml) and stirred 12 h. the residue obtained on concentration was subjected to chromatography on silica gel using a gradient of cma80 in ch2cl2 as the eluent . The product containing fractions were concentrated and subjected to chromatography on c18-reverse phase using a gradient from 40 to 60% aq ch3cn with 0.1% tfa . The product containing fractions were concentrated and again subjected to chromatography on silica gel using a gradient of cma80 in ch2cl2 to afford 13 free base . H nmr (300 mhz, cdcl3) 7.54 (s, 1h), 7.45 (d, j = 7.9 hz, 1h), 7.19 (d, j = 9.5 hz, 1h), 7.137.01 (m, 2h), 6.746.59 (m, 3h), 6.49 (bs, 1h), 6.02 (bs, 1h), 4.123.86 (m, 3h), 3.64 (t, j = 6.7 hz, 1h), 3.142.94 (m, 2h), 2.732.54 (m, 2h), 2.502.22 (m, 4h), 2.121.98 (m, 1h), 1.901.70 (m, 2h), 1.44 (d, j = 12.5 hz, 1h), 1.21 (s, 3h), 0.970.87 (m, 6h), 0.38 (d, j = 6.8 hz, 3h). C nmr (75 mhz, cdcl3) 172.4, 170.0, 156.5, 152.1, 138.8, 136.2, 130.8, 129.7, 129.3, 125.4, 125.1, 117.6, 113.2, 112.7, 60.6, 55.6, 54.9, 51.9, 50.9, 46.2, 38.8, 38.5, 31.1, 30.7, 30.2, 27.3, 19.6, 18.1, 16.1 . The free base was converted to 41.9 mg of the dihydrochloride salt (13%) as a white powder: mp 195200 c (fusion), []d + 103 (c 1.00, ch3oh). (c29h42cl2n4o33h2o) c, h, n. the amine 3-{(3r,4r)-1-[(2s)-2-amino-3-methylbutyl]-3,4-dimethylpiperidin-4-yl}benzamide (24 mg, 0.08 mmol) was added to a solution of the acid 36 (25 mg, 0.08 mmol), hbtu (30 mg, 0.08 mmol), and net3 (30 l, 0.2 mmol) in ch2cl2 (10 ml). After 12 h, the concentrated residue was subjected to chromatography on silica gel using a gradient up to 70% cma80 in ch2cl2 as the eluent . The product containing fractions were dissolved in ch2cl2 (5 ml) and tfa (5 ml) and stirred 12 h. the concentrated residue was subjected to chromatography on silica gel using a gradient of cma80 in ch2cl2 to afford 14 free base . H nmr (300 mhz, cdcl3) 7.72 (s, 1h), 7.54 (dd, j = 6.59, 14.69 hz, 3h), 7.297.46 (m, 2h), 7.017.22 (m, 2h), 5.906.78 (m, 4h), 3.99 (br s, 3h), 3.59 (d, j = 2.83 hz, 1h), 3.12 (d, j = 13.37 hz, 1h), 2.803.00 (m, 1h), 2.72 (d, j = 9.80 hz, 1h), 2.59 (d, j = 10.93 hz, 1h), 2.122.51 (m, 5h), 1.89 (dd, j = 6.97, 12.24 hz, 3h), 1.58 (d, j = 12.06 hz, 1h), 1.191.33 (m, 3h), 0.92 (t, j = 7.72 hz, 6h), 0.53 (d, j = 6.78 hz, 3h). C nmr (75 mhz, cdcl3) 172.2, 172.1, 170.2, 169.4, 151.1, 138.8, 136.2, 133.2, 131.1, 129.4, 128.3, 125.1, 125.0, 124.7, 124.1, 59.9, 55.9, 55.0, 51.3, 50.8, 46.9, 38.7, 38.5, 30.7, 30.7, 30.6, 27.4, 19.2, 17.9, 16.2 . Ms (esi) m / z 521.0 (m + h). The free base was converted to 20.4 mg (39%) of the dihydrochloride salt as a pale - yellow powder: mp 210215 c (fusion), []d + 101 (c 0.50, ch3oh). (c30h43cl2n5o33.25h2o) c, h, n. to a solution of 20 (100 mg, 0.3 mmol) in ch2cl2 (10 ml) was added 36 (100 mg, 0.3 mmol), hobt (10 mg, 0.1 mmol), and edchcl (75 mg, 0.4 mmol), followed by the addition of diisopropylethylamine (0.26 ml, 1.5 mmol). The resulting cloudy solution remained cloudy upon the addition of nmp (0.1 ml). After 12 h, the mixture was washed with saturated aqueous nahco3 (10 ml). The aqueous layer was extracted with ch2cl2:thf (2:1, 20 ml 2). The combined organic layers were washed with brine (5 ml), dried (na2so4), and concentrated . The resulting residue was purified by chromatography on silica gel using a gradient up to 40% cma80 in ch2cl2 as the eluent . The product containing fractions were combined and concentrated to afford 134 mg of the boc - protected intermediate . The intermediate was then dissolved in ch3oh (10 ml) to which aq hcl (6 n, 10 ml) was added . The resulting residue was subjected to chromatography on silica gel using a gradient up to 75% cma80 in ch2cl2 to afford 15 free base . H nmr (300 mhz, cdcl3) 7.387.51 (m, 2h), 6.957.28 (m, 9h), 5.936.34 (m, 2h), 3.844.04 (m, 2h), 3.46 (dd, j = 5.0, 10.3 hz, 1h), 3.11 (dd, j = 4.8, 17.1 hz, 1h), 2.622.83 (m, 2h), 2.55 (d, j = 10.7 hz, 1h), 2.302.46 (m, 3h), 2.082.29 (m, 2h), 1.751.99 (m, 2h), 1.51 (d, j = 12.2 hz, 1h), 1.131.28 (m, 3h), 0.740.94 (m, 6h), 0.56 (d, j = 6.8 hz, 3h). C nmr (75 mhz, cdcl3) 172.2, 169.3, 150.1, 138.8, 136.2, 131.1, 129.4, 128.0, 125.4, 125.3, 125.0, 59.5, 56.3, 55.2, 51.3, 50.6, 47.4, 38.6, 38.4, 31.0, 30.5, 27.5, 19.1, 17.7, 16.3 . Ms (esi) m / z 477.5 (m + h). The free base was converted to the dihydrochloride salt, which was sonicated in etoac . The solvent was decanted and the solids dried under nitrogen to afford 43 mg (24% over two steps) as a white powder: mp 218222 c (fusion), []d + 105 (c 0.195, ch3oh). (c29h42cl2n4o22.75h2o) c, h, n. the title compound was prepared by the addition of trifluoromethanesulfonic anhydride (3.4 ml, 20 mmol) to 3-[(3r,4r)-34-dimethylpiperidin-4-yl]phenol (16) (1.0 g, 4.9 mmol) and diisopropylethylamine (5.1 ml, 29 mmol) in ch2cl2 (30 ml) at 78 c . The solution was allowed to warm to room temperature, quenched with a brine wash, and concentrated . The ether layer was washed with 1 m hcl, aq nahco3, then brine . After drying (na2so4) h nmr (300 mhz, cdcl3) 7.417.48 (m, 1h), 7.247.32 (m, 1h), 7.117.19 (m, 2h), 4.01 (d, j = 13.2 hz, 1h), 3.543.71 (m, 2h), 3.313.45 (m, 1h), 2.36 (dt, j = 5.0, 13.1 hz, 1h), 2.062.20 (m, 1h), 1.74 (d, j = 13.6 hz, 1h), 1.42 (s, 3h), 0.75 (d, j = 7.0 hz, 3h). A solution of the triflate 17 (2.3 g, 4.9 mmol) in dmf (10 ml) was treated with nbu3 (3.5 ml, 15 mmol), pdcl2(pph3) (170 mg, 0.25 mmol), and formic acid (0.4 ml, 11 mmol). The solution was heated to 80 c for 5 h, then concentrated and purified by rapid elution of the product through silica gel using 20% etoac in hexanes as eluent to afford 1.43 g (90%) of 18 . H nmr (300 mhz, cdcl3) 7.307.39 (m, 2h), 7.207.28 (m, 3h), 3.98 (d, j = 13.0 hz, 1h), 3.62 (bs, 2h), 3.303.46 (m, 1h), 2.38 (dt, j = 5.1, 13.1 hz, 1h), 2.082.22 (m, 1h), 1.681.79 (m, 1h), 1.40 (s, 3h), 0.75 (d, j = 7.0 hz, 3h). A sample of triflamide 18 (520 mg, 1.6 mmol) was dissolved in toluene (10 ml) and thf (5 ml) and treated with lialh4 (320 mg, 8.3 mmol) and heated with a microwave to 150 c in a sealed tube for 10 min . The cooled solution was diluted with ether, chilled in an ice bath, and quenched with the sequential addition of water (0.3 ml), 15% naoh (0.3 ml), then water (0.6 ml). The resulting suspension was filtered through celite and concentrated to afford 226 mg of an oil . H nmr (300 mhz, cdcl3) 7.087.38 (m, 5h), 3.26 (dd, j = 3.3, 13.1 hz, 1h), 2.913.07 (m, 2h), 2.672.78 (m, 1h), 2.072.23 (m, 1h), 1.832.00 (m, 2h), 1.491.62 (m, 1h), 1.39 (s, 3h), 0.71 (d, j = 7.2 hz, 3h). The amine (19) (226 mg, 0.92 mmol) was combined with boc - l - valinal (355 mg, 1.8 mmol) in trifluoroethanol (5 ml) and treated with na(cn)bh3 (3 ml, 1 m in thf). After 1 h, the solution was concentrated and subjected to chromatography on silica gel using a gradient of etoac in hexanes as the eluent to afford 367 mg (61% over two steps from 18) of the boc - protected product . The concentrated residue was subjected to chromatography on silica gel eluting with a gradient of cma80 in ch2cl2 as eluent to afford 20 in quantitative yield . H nmr (300 mhz, cdcl3) 7.237.36 (m, 4h), 7.127.21 (m, 1h), 2.122.81 (m, 7h), 2.02 (d, j = 6.8 hz, 1h), 1.451.79 (m, 5h), 1.32 (s, 3h), 0.850.97 (m, 6h), 0.690.79 (m, 3h). This material was used without further purification . A solution of 3-{(3r,4r)-1-[(2s)-2-amino-3-methylbutyl]-3,4-dimethylpiperidin-4-yl}phenol (21) (6.84 g, 23.6 mmol) in chcl3 (230 ml) was refluxed with phthalic anhydride (4.5 g, 30 mmol) for 24 h. the cooled solution was washed with aq nahco3 then concentrated . The residue was subjected to chromatography on silica gel using a gradient of cma80 in ch2cl2 as the eluent to afford 5.80 g (58%) of 22 . H nmr (300 mhz, cdcl3) 7.74 (d, j = 2.3 hz, 2h), 7.63 (d, j = 2.6 hz, 2h), 7.08 (t, j = 7.8 hz, 1h), 6.70 (d, j = 8.1 hz, 1h), 6.64 (bs, 1h), 6.58 (d, j = 7.9 hz, 1h), 4.004.12 (m, 1h), 3.26 (5, j = 12.0 hz, 1h), 2.63 (d, j = 10.0 hz, 2h), 2.232.57 (m, 4h), 1.922.09 (m, 1h), 1.82 (d, j = 6.2 hz, 1h), 1.42 (d, j = 12.6 hz, 1h), 1.23 (s, 3h), 1.05 (d, j = 6.8 hz, 3h), 0.88 (d, j = 6.6 hz, 3h), 0.29 (d, j = 6.8 hz, 3h). Ms (esl) m / z 421.7 (m + h). The phthalimide - protected 22 (5.80 g, 13.8 mmol) was dissolved in ch2cl2 (150 ml) containing triethylamine (2.8 ml, 20 mmol) and n - phenyl - bis(trifluoromethanesulfonimide) (5.4 g, 15 mmol). After 12 h, the solution was washed with aq nahco3, dried (na2so4), and concentrated . The residue was subjected to chromatography on silica gel using a gradient 020% etoac in hexanes as the eluent to afford 4.81 g (63%) of triflate 23 . H nmr (300 mhz, cdcl3) 7.76 (bs, 2h), 7.617.70 (m, 2h), 7.247.35 (m, 1h), 7.16 (d, j = 7.9 hz, 1h), 6.977.05 (m, 2h), 3.984.13 (m, 1h), 3.26 (t, j = 12.2 hz, 1h), 2.592.76 (m, 2h), 2.242.57 (m, 4h), 2.02 (dt, j = 4.7, 12.4 hz, 1h), 1.84 (d, j = 5.8 hz, 1h), 1.25 (s, 3h), 1.05 (d, j = 6.6 hz, 3h), 0.89 (d, j = 6.8 hz, 3h), 0.26 (d, j = 7.0 hz, 3h). The triflate 23 (1.59 g, 2.9 mmol), benzylamine (0.46 ml, 4.2 mmol), potassium tert - butoxide (550 mg, 4.9 mmol), pd(oac)2 (7.7 mg, 0.035 mmol), and (2-biphenyl)di - tert - butylphosphine (21 mg, 0.07 mmol) were combined and degassed in toluene (3.5 ml) then stirred overnight at room temperature . The organic layer was washed with aq nh4cl, dried (na2so4), and concentrated . The residue was subjected to chromatography on silica gel using a gradient of etoac in hexanes as the eluent to afford 352 mg (24%) of 24 . H nmr (300 mhz, cdcl3) 7.75 (d, j = 2.6 hz, 2h), 7.64 (dd, j = 3.0, 5.3 hz, 2h), 7.207.37 (m, 5h), 7.03 (t, j = 7.8 hz, 1h), 6.52 (d, j = 7.7 hz, 1 h), 6.356.46 (m, 2h), 4.25 (s, 2h), 3.984.10 (m, 1h), 3.173.29 (m, 1h), 2.552.70 (m, 2h), 2.232.55 (m, 5h), 1.98 (dt, j = 4.5, 12.6 hz, 1h), 1.79 (d, j = 6.8 hz, 1h), 1.39 (d, j = 13.0 hz, 1h), 1.20 (s, 3h), 1.04 (d, j = 6.6 hz, 3h), 0.89 (d, j = 6.8 hz, 3h), 0.28 (d, j = 7.0 hz, 3h). The benzyl aniline 24 (353 mg, 0.69 mmol) was dissolved in etoh (100 ml) with 20% pd(oh)2/c (0.2 g) and shaken under hydrogen (45 psi) overnight . The filtered concentrate was subjected to chromatography on silica gel using 20% etoac in hexanes as the eluent to recover 202 mg of starting material, followed by 50% etoac in hexanes with 1% nh3 as the eluent to afford 55 mg (19%) of the desired aniline 25 . H nmr (300 mhz, cdcl3) 7.75 (d, j = 2.6 hz, 2h), 7.577.68 (m, 2h), 7.00 (t, j = 7.8 hz, 1h), 6.56 (d, j = 8.1 hz, 1 h), 6.396.50 (m, 2h), 4.06 (ddd, j = 4.8, 9.8, 11.6 hz, 1h), 3.24 (t, j = 12.1 hz, 1h), 2.562.70 (m, 2h), 2.212.54 (m, 4h), 1.922.03 (m, 1h), 1.751.87 (m, 1h), 1.40 (dd, j = 1.2, 12.9 hz, 1h), 1.21 (s, 3h), 1.05 (d, j = 6.6 hz, 3h), 0.850.94 (m, 3h), 0.29 (d, j = 67.0 hz, 3h). This material was used without further purification . A solution of 25 (1.34 g, 3.2 mmol) in dioxane (6 ml) and hcl (6 m, 6 ml) was stirred at reflux for 18 h. the mixture was concentrated then partitioned between aq nahco3 and etoac . The organic layer was separated, dried (na2so4), and concentrated . The resulting residue was subjected to chromatography on silica gel with 50% etoac in cma80 as the eluent (rf 0.5) to afford 0.41 g (44%) of 26 as a light - orange oil . H nmr (cdcl3) 7.10 (t, j = 7.8 hz, 1h), 6.676.73 (m, 1h), 6.62 (t, j = 2.0 hz, 1h), 6.51 (ddd, j = 0.9, 2.3, 7.8 hz, 1h), 3.61 (br s, 1h), 2.572.78 (m, 2h), 2.51 (dt, j = 2.6, 11.8 hz, 1h), 2.272.43 (m, 3h), 2.25 (d, j = 3.6 hz, 1h), 2.132.22 (m, 1h), 1.892.03 (m, 2h), 1.68 (br s, 2h), 1.451.59 (m, 2h), 1.271.31 (m, 3h), 0.92 (d, j = 7.0 hz, 6h), 0.78 (d, j = 7.0 hz, 3h). Ms (esi) m / z 290.3 (m + h). This material was used without further purification . The aniline 25 (55 mg, 0.13 mmol) was dissolved in ch2cl2 (1 ml) and treated with bf3oet2 (32 l, 0.26 mmol) then isoamyl nitrite (26 l, 0.20 mmol). After stirring 15 min, the resulting crystalline solids were collected by filtration, dried, and heated neat . The resulting residue was subjected to chromatography on silica gel eluting with etoac as the eluent to afford 36 mg (66%) of 27 . H nmr (cdcl3) 7.797.90 (m, 2h), 7.687.79 (m, 2h), 7.117.34 (m, 2h), 6.807.04 (m, 3h), 4.144.26 (m, 1h), 4.00 (t, j = 11.9 hz, 1h), 3.243.78 (m, 4h), 2.55 (br s, 2h), 2.182.37 (m, 2h), 1.41 (s, 3h), 1.061.17 (m, 3h), 0.770.89 (m, 3h), 0.520.74 (m, 3h). This material was used without further purification . A solution of 25 (340 mg, 0.81 mmol) in aq hcl (37%, 5 ml) was cooled to 5 c and stirred for 10 min . A solution of nano2 (63 mg, 0.89 mmol) in water (1.5 ml) was added dropwise to the reaction which then was stirred for 1 h. a cold solution of copper(i) chloride (92 mg, 0.93 mmol) in water (1.5 ml) was then added dropwise . The reaction mixture was stirred for 30 min and allowed to warm to rt then was heated to 65 c for 3 h. the resulting suspension was poured in to a mixture of concd nh4oh (20 ml) and ice (6 g). The resulting solution with was extracted with ethyl acetate (2 40 ml). The combined organic layers were dried (na2so4) and evaporated to a residue, which was subjected to chromatography on silica gel using a gradient up to 50% etoac in hexanes as the eluent to afford 100 mg (28%) of 28 as an oil that solidified to a white solid upon standing . H nmr (cdcl3) 7.77 (br s, 2h), 7.65 (br s, 2h), 6.957.21 (m, 4h), 4.06 (m, 1h), 3.26 (t, j = 12.1 hz, 1h), 2.582.79 (m, 2h), 2.182.56 (m, 3h), 1.932.11 (m, 1h), 1.761.91 (m, 1h), 1.371.50 (m, 1h), 1.24 (s, 3h), 1.06 (d, j = 6.8 hz, 3h), 0.90 (d, j = 6.8 hz, 3h), 0.28 (d, j = 6.9 hz, 3h). C nmr (cdcl3) 169.1, 152.8, 134.0, 133.6, 132.0, 129.2, 125.8, 125.3, 123.7, 122.8, 57.2, 55.0, 54.1, 51.4, 38.6, 38.5, 30.4, 29.9, 27.2, 20.4, 20.3, 15.4 . A solution of 25 (250 mg, 0.6 mmol in aq hbr (48%, 0.5 ml) and water (1 ml) was cooled to 5 c and stirred for 10 min . A solution of nano2 (46 mg, 0.65 mmol) in water (1 ml) was added dropwise, and the reaction mixture stirred for 2 h. urea was added to the reaction mixture to consume any excess nitrous acid . Copper(i) bromide (103 mg, 0.72 mmol), aq hbr (48%, 0.2 ml), and water (0.5 ml) were added consecutively to the reaction mixture . After stirring for 1 h, the resulting suspension was poured in to a mixture of concd nh4oh (20 ml) and ice (6 g). The resulting solution was extracted with ethyl acetate (2 40 ml). The combined organic layers were dried (na2so4) and evaporated to a residue, which was subjected to chromatography on silica gel using a gradient up to 50% etoac in hexanes as the eluent to afford 110 mg (38%) of 29 as a colorless oil . H nmr (cdcl3) 7.77 (br s, 2h), 7.65 (dd, j = 2.9, 5.2 hz, 2h), 7.187.26 (m, 2h), 6.947.18 (m, 2h), 3.944.21 (m, 1h), 3.26 (t, j = 12.1 hz, 1h), 2.582.78 (m, 2h), 2.202.57 (m, 4h), 1.942.03 (m, 1h), 1.771.93 (m, 1h), 1.371.55 (m, 1h), 1.23 (s, 3h), 1.06 (d, j = 6.6 hz, 3h), 0.91 (d, j = 6.8 hz, 3h), 0.28 (d, 3h, j = 7.2 hz, 3h). C nmr (cdcl3) 169.1, 153.1, 133.6, 132.0, 129.6, 128.7, 128.3, 128.0, 125.4, 125.1, 124.2, 122.8, 122.5, 57.2, 55.0, 54.1, 51.4, 38.7, 38.4, 30.3, 29.9, 27.3, 20.4, 20.3, 15.5 . A solution of 27 (36 mg, 0.085 mmol) was heated at reflux in etoh (10 ml) and hydrazine (1 ml) overnight . The concentrated residue was dissolved in etoac then washed with aq nahco3 . The organic layer was dried (na2so4) and concentrated to afford 24 mg (96%) of the amine 30 . H nmr (300 mhz, cdcl3) 7.127.26 (m, 1h), 6.98 (d, j = 8.1 hz, 1h), 6.90 (dd, j = 1.9, 11.3 hz, 1h), 6.79 (dt, j = 2.1, 8.3 hz, 1h), 2.072.77 (m, 8h), 1.831.96 (m, 1h), 1.421.58 (m, 2h), 1.081.31 (m, 5h), 0.740.95 (m, 6h), 0.600.74 (m, 3h). This material was used without further purification . To a solution of 28 (100 mg, 0.23 mmol) in ethanol (15 ml) was added hydrazine monohydrate (115 mg, 2.3 mmol). The reaction mixture was stirred at reflux under nitrogen for 12 h and then concentrated to obtain a white solid that was dissolved in aq nahco3 (10 ml) and extracted with etoac (2 20 ml). The combined organic layers were dried (na2so4) and concentrated to afford 56 mg (79%) of 31 as a colorless oil . H nmr (300 mhz, cdcl3) 6.947.18 (m, 4h), 2.502.73 (m, 3h), 2.45 (m, 1h), 2.042.38 (m, 3h), 1.792.02 (m, 2h), 1.371.56 (m, 2h), 1.23 (m, 3h), 0.85 (d, j = 6.8 hz, 6h), 0.69 (d, j = 6.9 hz, 3h). C nmr (75 mhz, cdcl3) 151.7, 133.1, 128.3, 124.9, 124.5, 122.8, 61.7, 53.5, 51.9, 51.0, 37.9, 37.6, 31.1, 29.6, 26.4, 18.3, 17.2, 15.2 . Ms (esi) m / z 309.4 (m + h). This material was used without further purification . To a solution of 29 (110 mg, 0.23 mmol) in ethanol (15 ml) was added hydrazine monohydrate (115 mg, 2.3 mmol). The reaction mixture was stirred at reflux under nitrogen for 12 h and then concentrated to obtain a white solid that was dissolved in aq nahco3 (10 ml) and extracted with etoac (2 20 ml). The combined organic layers were dried (na2so4) and concentrated to obtain 94 mg (99%) of 32 as a colorless oil . H nmr (300 mhz, cdcl3) 7.33 (s, 1h), 7.177.28 (m, 1h), 7.037.17 (m, 2h), 2.512.71 (m, 3h), 2.102.49 (m, 4h), 1.822.00 (m, 1h), 1.381.65 (m, 3h), 1.23 (m, 3h), 0.85 (d, j = 6.8 hz, 6h), 0.560.76 (m, 3h). C nmr (75 mhz, cdcl3) 152.1, 128.7, 127.8, 127.4, 127.1, 124.5, 124.3, 123.2, 121.6, 61.8, 53.5, 51.9, 51.0, 37.9, 37.6, 31.2, 29.6, 26.4, 18.3, 17.2, 15.3 . Ms (esi) m / z 353.4 (m + h). A solution of boc-7-hydroxy - d - tic (1.47 g, 5 mmol) in toluene (35 ml) and ch3oh (10 ml) was treated with a solution of tmschn2 in ether (2.0 m, 2.5 ml) until a slight yellow persisted . The residue was dissolved in ch2cl2 (50 ml) and net3 (0.9 ml, 6.5 mmol) and treated with tf2o (0.85 ml, 5.0 mmol) at 0 c . The reaction was allowed to warm to room temperature and concentrated to a residue and subjected to a plug of silica gel, eluting with 20% etoac in hexanes as the eluent . The fractions containing product were concentrated and dissolved in dmf (6 ml) with zn(cn)2 (1.0 g, 8.5 mmol). The mixture was degassed and kept under nitrogen as pd(pph3)4 (200 mg, 0.2 mmol) was added . The mixture was heated to 100 c for 4 h, cooled, then partitioned between etoac and aq nahco3 . The organic layer was dried (na2so4), concentrated, and subjected to chromatography on silica gel using 20% etoac in hexanes to afford 1.37 g of 35 (86% over 3 steps). H nmr (300 mhz, cdcl3) 7.397.52 (m, 2h), 7.227.31 (m, 1h), 4.684.81 (m, 1h), 4.424.60 (m, 1h), 3.64 (d, j = 4.9 hz, 3h), 3.30 (d, j = 2.1 hz, 1h), 3.22 (d, j = 5.8 hz, 2h), 1.50 (d, j = 19 hz, 9h). This material was used without further purification . A sample of 35 (320 mg, 1.0 mmol) was dissolved in dioxane (2 ml) and thf (1 ml) then treated with aq lioh (1 m, 3 ml) overnight . The resulting solution was cooled in an ice bath and treated cautiously with h2o2 (30%, 1 ml). After warming, the reaction mixture was acidified with hcl (2 m) and diluted with water . The resulting solids were separated by filtration and dried to afford 230 mg of 36 (72%). H nmr (300 mhz, dmso - d6) 12.72 (s, 1h), 7.90 (br s, 1h), 7.617.74 (m, 1h), 7.217.37 (m, 1h), 4.89 (br s, 1h), 4.68 (s, 1h), 4.554.64 (m, 1h), 4.52 (d, j = 5.7 hz, 1h), 4.364.48 (m, 1h), 3.063.26 (m, 2h), 1.331.53 (m, 9h). This material was used without further purification . The binding assays were performed for 60 min using 4 g herg expressing membranes, 3 nm [h]astemizole, and various concentrations of the test agent in a binding buffer (10 mm hepes, ph 7.4, 130 mm nacl, 5 mm kcl, 0.8 mm mgcl2, 1 mm naedta, 10 mm glucose, 0.1% bsa). Binding was terminated by rapid filtration onto gf / b fiber filtermats, presoaked in 0.3% polyethylenimine, followed by rapid washing 6 times (2 ml) with ice - cold solution containing 25 mm tris - hcl, ph 7.4, 130 mm nacl, 5 mm kcl, 0.8 mm mgcl2, 0.05 mm cacl2, and 0.1% bsa using a brandel harvester . Data were analyzed using nonlinear regression (graphpad prism), and ki values were determined as described before . All experiments were performed at least twice in duplicate, and data reported are mean values . For these experiments, 10 mm dmso stocks of compounds were directly diluted into 10 mm phosphate buffer at ph 7.4 or 3 and shaken for 90 min at room temperature . After the incubation, samples were filtered through a 0.4 m filterplate (millipore). Analysis of compounds was performed by lc / ms using previously available methods and concentrations determined . A commercially available pampa assay system was used (bd gentest precoated pampa system). Assays were performed in duplicate at 10 m final concentration at ph 7.4 and 5.5 as has been described previously in pbs buffer . The ligand preparation, receptor preparation, and docking calculations were conducted under our previously reported methods.
Various strategies have been widely investigated to enhance the bioavailability of poorly absorbed drugs in order to increase their clinical efficacy when administered orally . It is estimated that between 40% and 70% of all new chemical entities identified in drug discovery programs are insufficiently soluble in aqueous media [1, 2]. The increase in the proportion of poorly soluble candidates is frequently attributed to improvements in synthesis technology, which has enabled the design of very complicated compounds, and a change in discovery strategy from a so - called phenotypic approach to a target - based approach . Various physicochemical properties which contribute to the poor solubility of various drugs include their complex structure, size, high molecular weight, high lipophilicity, compound h - bonding to solvent, intramolecular h - bonding, intermolecular h - bonding (crystal packing), crystallinity, polymorphic forms, ionic charge status, ph, and salt form . Lipinski's rule of five has been widely proposed as a qualitative predictive model for the assessment of absorption of poorly absorbed compounds . In the discovery the rule of 5 predicts that poor absorption or permeation is more likely when there are more than 5 h - bond donors, 10 h - bond acceptors, the molecular weight is greater than 500, and the calculated log p is greater than 5 . The rule of five only holds for compounds that are not substrates for active transporters and efflux mechanisms . Thus, in vivo assessment of new drug candidates in animal model is performed to assess the absorption of drug . Poorly absorbed drugs pose a challenge to the formulation scientists to develop suitable dosage form which can enhance their bioavailability . Broadly, poorly soluble drugs can be formulated in three different forms to overcome the challenge of poor absorption crystalline solid formulations, amorphous formulations, and lipid formulations . Modification of the physicochemical properties such as salt formation and micronization of the crystalline compound to increase the surface area and thus dissolution may be one approach to improve the dissolution rate of the drug . Particle size of about 25 m can be achieved by micronization using air - jet mill . The nanocrystal technology can reduce the crystalline particle size to 100250 nm using ball - milling, dense gas technologies, and so forth . However, these methods have their own limitations . For instance, salt formation of neutral compounds is not feasible . Particle size reduction may not be desirable in situations where poor wettability and handling difficulties are experienced for very fine powders . Amorphous formulations include solid solutions which can be formed using a variety of technologies including spray drying and melt extrusion [911]. Other formulation strategies which are most popularly adopted to enhance the bioavailability of such drugs include the complexation with cyclodextrins, formulation of polymeric conjugates, nanoparticles, solid lipid nanoparticles (sln), use of permeation enhancers, and surfactants . In recent years the most popular approach is the incorporation of the active poorly water soluble component into inert lipid vehicles such as oils, surfactant dispersions, solid dispersions, solid lipid nanoparticles, emulsions, microemulsions, nanoemulsions, self - emulsifying formulations (sef), micro / nanoemulsifying formulations, and liposomes . Table 1 provides a brief indication of the main formulation strategies and the main advantages and disadvantages of each approach . Lipid formulations for oral administration of drugs are a diverse group of formulations having a wide range of properties . The utility of solubilizing lipid - based formulations for improving the gastrointestinal (gi) absorption of poorly water - soluble, hydrophobic drugs is well documented in the literature . These generally consist of a drug dissolved in a blend of excipients (5 classes of excipients) with wide variety of physicochemical properties ranging from pure triglyceride oils, mono- and diglycerides, and substantial proportion of lipophilic or hydrophilic surfactants and cosolvents . The primary mechanism of action by which a lipid formulation leads to improved bioavailability is usually avoidance of the slow dissolution process which limits the bioavailability of hydrophobic drugs from solid dosage forms . Preferably the formulation allows the drug to remain in a dissolved state throughout its transit in the git . The drug for absorption can be enhanced by formulation of the drug as a solubilizate within a colloidal dispersion . This objective can be achieved by formulation of the drug in a self - emulsifying system . Among various approaches, the self - emulsifying drug delivery system has gained more attention due to enhanced oral bioavailability enabling reduction in dose, more consistent temporal profiles of drug absorption, selective targeting of drug(s) toward specific absorption window in git, and protection of drug(s) from the hostile environment in gut . Seddss, smeddss and sneddss are physically stable isotropic mixtures of oil, surfactant, cosurfactant, and solubilized drug substance that rapidly and spontaneously form fine oil in water emulsions, microemulsions, or nanoemulsions, respectively, when introduced into aqueous phases under gentle agitation . Thus, self - emulsifying formulations are readily dispersed in the gi tract, where the motility of the stomach and small intestine provides the agitation necessary for emulsification . The potential advantages of the self - emulsifying systems include 100% drug entrapment capacity, physically stable formulation (can also be filled in capsules), no dissolution step required, formation of submicron droplet size, thus increasing absorption surface area, increase in rate and extent of absorption, and thus increased bioavailability . They also have potential for effective delivery of bcs class iii, bcs class iv and hydrolytically susceptible drugs . Seddss have been described as systems that produce emulsions with a droplet size between 100 and 300 nm while smeddss form transparent microemulsions with a droplet size of less than 50 nm . However, sedds generally refers to all types of self - emulsifying systems unless otherwise described, while sneddss describe systems which form nanoemulsions upon dispersion in aqueous media . When compared with emulsions, which are sensitive and metastable dispersed forms, these self - emulsifying formulations are physically stable, easily manufactured, and are suitable for oral delivery as unit dosage form in soft or hard gelatin capsules due to the anhydrous nature . Thus, for lipophilic drug compounds that exhibit dissolution rate - limited absorption, these systems may offer an improvement in the rate and extent of absorption and result in more reproducible blood - time profiles . Being anhydrous, these systems also offer a great potential for the formulation and administration of hydrolytically susceptible drugs . Sedds are also found to increase the intestinal permeability and minimize the effect of ph on drug absorption . While the primary mechanism by which these formulations are thought to improve drug absorption is through elimination of the need for preabsorptive drug solubilisation in the gastrointestinal tract (git), other mechanisms may include protection from chemical and enzymatic degradation localized in the aqueous environment of the git and promotion of lymphatic drug transport, which circumvents hepatic first - pass metabolism . Figure 1 gives the schematic diagram of intestinal drug transport from lipid - based formulations via the portal and the mesenteric lymphatic routes . The physicochemical characteristics of the drug substance, the lipid excipients themselves, and the dispersibility of the formulation in vivo will determine both the uptake of the drug in the git as well as the degree of participation of the portal venous and mesenteric lymphatic pathways in overall drug absorption . The main consideration in selecting appropriate excipients for any lipid - based formulation is in identifying an excipient or their combination having the ability to solubilise the entire drug dose in a volume acceptable for unit oral administration . Self - emulsification has been shown to be specific to the nature of the oil / surfactant pair; the surfactant concentration and oil / surfactant ratio; and the temperature at which self - emulsification occurs . In support of these facts, it has also been demonstrated that only very specific pharmaceutical excipient combinations could lead to efficient self - emulsifying systems . The drug must also be physically and chemically stable in the formulation and the drug release characteristics must remain constant during the shelf life of the formulation . The latter requirement is dependent on the physical and chemical stability of the excipients which must be carefully monitored during formulation development . Excipients published by usfda or from other inactive ingredients approved and published by regulatory agencies . The main excipients in a self - emulsifying system include the lipids (oils), surfactant, and cosurfactant . A few examples of various excipients used in different commercial products are given in table 3 . The oil represents one of the most important excipients in the self - emulsifying formulations because it can solubilise marked amounts of the lipophilic drug, facilitate self - emulsification, and increase the fraction of lipophilic drug transported via the intestinal lymphatic system, thereby increasing absorption from the gi tract depending on the molecular nature of the triglyceride [16, 46]. Despite the considerable potential that these lipid excipients offer, very few lipid based formulations have reached the pharmaceutical market place . This may be due to the insufficient information regarding the relatively complex physical chemistry of lipids and concerns about formulated drug chemical and physical stability . In addition to these concerns, the interaction of a lipid - based formulation with the gi environment and its impact on drug absorption is also important . For instance, digestible lipids have been shown to be considerably more efficient enhancers of poorly soluble drug absorption, as compared to nondigestible lipids (e.g., liquid paraffin). Both long and medium chain triglyceride oils with different degrees of saturation have been used for the design of self - emulsifying formulations . The edible oils which represent the logical and preferred lipid excipient choice for the development of sedds are not frequently selected due to their poor ability to dissolve large amounts of lipophilic drugs . Modified or hydrolyzed vegetable oils have been widely used since these excipients form good emulsification systems with a large number of surfactants approved for oral administration and exhibit better drug solubility properties . They offer formulative and physiological advantages, and their degradation products resemble the natural end products of intestinal digestion . Novel semisynthetic medium chain derivatives, which can be defined as amphiphilic compounds with surfactant properties, are progressively and effectively replacing the regular medium chain triglyceride oils in the self - emulsifying drug delivery systems . The lipids exert their effects possibly through several complex mechanisms that can lead to alteration in the biopharmaceutical properties of the drug, such as increased dissolution rate of the drug and solubility in the intestinal fluid, protection of the drug from chemical as well as enzymatic degradation in the oil droplets, and the formation of lipoproteins promoting the lymphatic transport of highly lipophilic drugs . The amount of lipid contained in a formulation also influences the drug absorption primarily via solubilization in the git and potentially through activation of gi lipid digestion resulting in increased secretion of pancreatic juice and bile . Thus, the impact of any lipid - based formulation on the lipid digestion processes must be considered, particularly when multiple dosage units of a lipid - based formulation are administered as a single dose, which is common for many anti - hiv drugs . The self - emulsifying properties require the incorporation of relatively large amounts of surfactant in the formulation in addition to the oily drug carrier vehicle . The surfactants may improve the affinity between lipids and intestinal membrane or increase the permeability of the intestinal membrane . Surfactants increase the permeability by partitioning into the cell membrane and disrupting the structural organization of the lipid bilayer leading to permeation enhancement . They also exert their absorption enhancing effects by increasing the dissolution rate of the drug . Several compounds exhibiting surfactant properties may be employed for the design of self - emulsifying systems, the most widely recommended ones being the nonionic surfactants with a relatively high hydrophilic - lipophilic balance (hlb) values [2, 51, 52]. Emulsifiers of natural origin (e.g., lecithin, akoline medium chain monoglycerides (mcm), and peceol) are normally preferred since they are considered to be safer than the synthetic surfactants . However, these excipients have limited self - emulsification efficiency . Various vegetable oil derivatives like acrosyl (castor oil derivative) are still being found to give optimum self - emulsification . Nonionic surfactants are normally preferred over their ionic counterparts due to more favourable safety profiles and greater emulsion stability over a wider range of ph and ionic strength . In addition, nonionic surfactants can produce reversible changes in intestinal mucosal permeability, further facilitating absorption of the coadministered drug . Hydrophobic surfactants can penetrate membranes causing changes in membrane fluidity and permeability . Generally single alkyl chains are more penetrative, so bulky surfactants such as polysorbates and triglyceride ethoxylates are found to be less toxic . Usually the surfactant concentration ranges between 30 and 60% of the total formulation in order to form stable sedds . It is very important to determine the surfactant concentration properly as large amounts of surfactants may cause gi irritation . However, the extremely small lipid droplet size produced by smedds and snedds formulations promotes rapid stomach emptying and wide dispersion throughout the git, minimizing exposure to high local surfactant concentrations and thus reducing the irritation potential . The surfactant involved in the formulation of sedds should have a relatively high hlb and hydrophilicity to enable rapid and facile dispersion in the aqueous gi fluid as a very fine oil - in - water emulsion, and hence good self - emulsifying performance the use of surfactant blends to achieve the hydrophilic - lipophilic balance (hlb) value required for emulsification has often been proven to provide superior self - emulsifying properties relative to the use of a single surfactant possessing the desired hlb . One or more cosolvents are often added to the formulation to assist in solubilising high concentrations of the drug . Surfactants are amphiphilic in nature and they can dissolve or solubilize relatively high amounts of hydrophobic drug compounds . There is a relationship between the droplet size and the concentration of the surfactant being used . In many cases, this could be explained by the stabilization of the oil droplets as a result of the localization of the surfactant molecules at the oil - water interface . On the other hand, in some cases this phenomenon could be attributed to the interfacial disruption elicited by enhanced water penetration into the oil droplets mediated by the increased surfactant concentration and leading to ejection of oil droplets into the aqueous phase . Attempts have been made to evaluate the toxicity of pharmaceutical excipients and sedds or smedds formulations in vitro in caco-2 cell monolayers [20, 56]. The production of an optimum self - emulsifying formulation requires relatively high concentrations (generally more than 30% w / w) of surfactants . The presence of the cosurfactants decreases the bending stress of interface and allows the interfacial film sufficient flexibility to take up different curvatures required to form nanoemulsions over a wide range of composition . Organic solvents such as, ethanol, propylene glycol (pg), and polyethylene glycol (peg) are suitable for oral delivery, and they enable the dissolution of large quantities of either the hydrophilic surfactant or the drug in the lipid base . On the other hand, alcohols and other volatile cosolvents have the disadvantage of evaporating into the shells of the soft gelatin or hard, sealed gelatin capsules in conventional self - emulsifying formulation leading to drug precipitation . Thus, alcohol - free formulations have also been designed, but their lipophilic drug dissolution ability may be limited . It has been suggested by reiss that self - emulsification takes place when the entropy change favouring dispersion is greater than the energy required to increase the surface area of the dispersion . The free energy of a conventional emulsion formulation is a direct function of the energy required to create a new surface between the two phases (oil and water phases) and can be described by (1)g=iniri2, where g is free energy associated with the process (ignoring the free energy of mixing), n is the number of droplets, r is radius of globules, and is the interfacial energy . The two phases of the emulsion tend to separate with time to reduce the interfacial area and thus the free energy of the systems . The conventional emulsifying agents stabilize emulsions resulting from aqueous dilution by forming a monolayer around the emulsion droplets, reducing the interfacial energy and forming a barrier to coalescence . In contrast, emulsification occurs spontaneously with self - emulsifying formulations because the free energy required to form the emulsion is either low and positive or negative [17, 49]. It is necessary for the interfacial structure to show no resistance against surface shearing in order for emulsification to take place . Figure 2 depicts the schematic presentation of the mechanism happening during addition of water in sedds in a simplified way . The ease of emulsification was suggested to be related to the ease of water penetration into the various liquid crystal (lc) or gel phases formed on the surface of the droplet . The interface between the oil and aqueous continuous phases is formed upon addition of a binary mixture (oil / nonionic surfactant) to water . This is followed by the solubilisation of water within the oil phase as a result of aqueous penetration through the interface . Eventually, everything that is in close proximity with the interface will be lc, the actual amount of which depends on the surfactant concentration in the binary mixture . Thus, following gentle agitation of the self - emulsifying system, water will rapidly penetrate into the aqueous cores and lead to interface disruption and droplet formation . As a consequence of the lc interface formation surrounding the oil droplets, self - emulsifying formulations become very stable to coalescence . Detailed studies have also been carried out to determine the involvement of the lc phase in the emulsion formation process [31, 33, 59]. Also, particle size analysis and low frequency dielectric spectroscopy (lfds) were utilized to examine the self - emulsifying properties of a series of imwitor 742 (a mixture of mono- and diglycerides of capric and caprylic acids)/tween 80 systems . The results suggested that there might be a complex relationship between lc formation and emulsion formation . Moreover, the presence of the drug compound may alter the emulsion characteristics, probably by interacting with the lc phase . Nevertheless, the correlation between the lc formation and spontaneous emulsification has still not been established . The self - emulsifying drug delivery systems offers advantages in addressing the challenges of drug solubility and absorption; the next challenge remains the delivery of the drug in an acceptable dosage form . The oral dosage forms are the preferred drug administration route, and lipid formulations offer flexibility for oral dosage forms because they can be formulated as solutions, semisolid, and solid forms . Conventional self - emulsifying drug delivery systems, however, are mostly prepared in a liquid form, which can produce some disadvantages, for example, low stability, irreversible drugs / excipients precipitation, large volume of dose, difficulty in handling and portability, and few choices of dosage forms . To address these problems, such systems require the solidification of liquid self - emulsifying systems into powders to produce various solid dosage forms (se capsules, se tablets, se pellets, se beads, and so on). The liquid sedds can be converted into solid dosage form without affecting drug release property . Thus, s - seddss combine the advantages of sedds (i.e., enhanced solubility and bioavailability) with those of solid dosage forms (e.g., high stability and reproducibility, compact dosage form, ease of handling and portability, and better patient compliance). Knowing the advantages of solid dosage forms, s - seddss have been extensively investigated in recent years, as they frequently correspond to more effective alternatives to conventional liquid sedds . Examples include the development of s - sedds of dexibuprofen, nimodipine, and hydrochlorothiazide . From the perspective of dosage forms, s - seddss focus on the incorporation of liquid / semisolid se ingredients into powders / nanoparticles by different solidification techniques . The concept of super - snedds of poorly soluble drug simvastatin has also been investigated . Super - sneddss (200% drug - loaded) were produced by subjecting the snedds preconcentrates to a heating and cooling cycle . The relative bioavailability of the drug from super - seddds was found to increase significantly (180 53.3%) compared to conventional snedds . The supersaturatable sedds was designed, using a small quantity of hpmc (hydroxy propyl methyl cellulose or other polymers) in the formulation to prevent precipitation of the drug by generating and maintaining a supersaturated state in vivo . This system contained a reduced amount of a surfactant, thereby minimizing gi side effects and . For enhancing oral bioavailability of drugs with high solubility and low permeability, water - in - oil - in - water (w / o / w) double emulsions are also investigated . A novel formulation, self - double - emulsifying drug delivery systems (sdeddss) were formulated by mixing of hydrophilic surfactants and water - in - oil (w / o) emulsions . Sdedds can spontaneously emulsify to w / o / w double emulsions in the mixed aqueous gastrointestinal environment, with drugs encapsulated in the internal water phase of the double emulsions . Solid seddss are being developed from liquid / semisolid sedds mainly by adsorption on solid carriers, spray drying, lyophilization, melt extrusion, and nanoparticle technology . Such powders / nanoparticles, which are referred to as se nanoparticles / dry emulsions / solid dispersions, are usually further processed into other solid se dosage forms or, alternatively, filled into capsules (i.e., se capsules). Se capsules also include those capsules into which liquid / semisolid seddss are directly filled without any solidifying excipient . Other solid se dosage forms that have emerged in recent years include se pellets / tablets, se microspheres / nanoparticles, and se suppositories / implants . Free flowing powders may be obtained from liquid se formulations by adsorption on solid carriers . The adsorption process is simple and just involves addition of the liquid formulation onto inert carriers and mixing them in a blender . The resulting powder may then be filled directly into capsules or, alternatively, mixed with suitable excipients before compression into tablets . Sedds can be adsorbed at high levels (up to 70% w / w) onto suitable carriers . Solid carriers can be microporous inorganic substances, high surface - area colloidal inorganic adsorbent substances, cross - linked polymers, or nanoparticle adsorbents, for example, silica, silicates, magnesium trisilicate, magnesium aluminium silicate (neusilin) microporous calcium silicate (florite tm re) magnesium hydroxide, talcum, crospovidone, cross - linked sodium carboxymethyl cellulose, and cross - linked polymethyl methacrylate . The self - emulsifying powder was prepared by adsorbing the liquid sedds onto neusilin as carrier to improve the solubility of poorly soluble lercanidipine hydrochloride . Nanoparticle adsorbents comprise porous silicon dioxide, carbon nanotubes, carbon nanohorns, charcoal, and so forth . In this technique, the liquid sedds is added to a solution of suitable solid carrier with stirring to obtain the o / w emulsion . This is then atomized into a spray of droplets in a drying chamber, where the volatile phase (e.g., the water contained in an emulsion) evaporates, forming dry particles under controlled temperature and airflow conditions [36, 45]. Such particles can be further prepared into tablets or capsules . The atomizer, the temperature, the most suitable airflow pattern, and the drying chamber design are selected according to the drying characteristics of the product and powder specification . Lyophilization or freeze - drying involves transfer of heat and mass to and from the product under preparation . Freeze drying of an oil - in - water emulsion can be an alternative method for the production of dry emulsions . Lyophilization has been thought as a molecular mixing technique where the drug and carrier are codissolved in a common solvent, frozen, and sublimed to obtain a lyophilized molecular dispersion . The potential applications of lyophilization in manufacturing of solid dispersions have successfully been investigated [65, 69, 70]. A slow cooling rate and addition of amorphous cryoprotectants has been reported to have the best stabilizing effects during lyophilization of oil - in - water emulsions . Maltodextrins are also useful matrix forming agent in the formulation of freeze - dried tablets . Melt granulation is a technique in which powder agglomeration is obtained through the addition of a lipid as binder that melts or softens at relatively low temperatures . Melt granulation offers several advantages over the conventional wet granulation, since the liquid addition and the subsequent drying phase are omitted . The main parameters that control the granulation process are impeller speed, mixing time, binder particle size, and the viscosity of the binder . For example, gelucires, a family of vehicles derived from the mixtures of mono-/di-/triglycerides and polyethylene glycols (peg) esters of fatty acids, is able to increase the dissolution rate compared with peg usually used before, probably owing to its se property . Other lipid - based excipients evaluated for melt granulation to create solid ses include lecithin, partial glycerides, or polysorbates . In all cases, melt extrusion is a solvent - free process that allows high drug loading (60%), as well as content uniformity . Extrusion is a procedure of converting a raw material with plastic properties into a product of uniform shape and density, by forcing it through a die under controlled temperature, product flow, and pressure conditions . The size of the extruder aperture will determine the approximate size of the resulting spheroids . The extrusion - spheronization process is commonly used in the pharmaceutical industry to make uniform sized spheroids (pellets). The extrusion - spheronization process requires the following steps: dry mixing of the active ingredients and excipients to achieve a homogenous powder; wet massing with binder; extrusion into rope - like extrudate; spheronization from the extrudate to spheroids of uniform size; drying; sifting to achieve the desired size distribution and coating [34, 66]. Examples of such problems include the following.amount of solidifying excipients may affect the release of the drug.nature of the excipients used may affect the drug absorption.probability of irreversible phase separation on reconstitution.clogging of spray nozzles due to oil content in spray - drying method.degradation of drug during solidification process.reduction in drug loading capacity.difficulty in ensuring content uniformity.probability of residual solvents used during granulation . Probability of residual solvents used during granulation . In order to reduce the amount of solidifying excipients required for transformation of sedds into solid dosage forms colloidal silicon dioxide (aerosil 200) was selected as a gelling agent for the oil - based systems, which served the dual purpose of reducing the amount of required solidifying excipients and aiding in slowing down of the drug release .after administration of capsules containing conventional liquid se formulations, emulsion droplets form and subsequently disperse in the gi tract to reach sites of absorption . However, if irreversible phase separation of the emulsion occurs, an improvement of drug absorption cannot be expected . For handling this problem, sodium dodecyl sulfate was added into the se formulation .with the similar purpose, the supersaturatable sedds was designed, using a small quantity of hpmc (or other polymers) in the formulation to prevent precipitation of the drug by generating and maintaining a supersaturated state in vivo . This system contains a reduced amount of a surfactant, thereby minimizing gi side effects . These excipients have the potential to increase the absorption of poorly water - soluble drugs relative to previously used peg solid dispersions and may also be filled directly into hard gelatin capsules in the molten state, thus obviating the former requirement for milling and blending before filling . Se excipients like gelucire1 44/14, gelucire150/02, labrasol1, transcutol1, and tocopheryl polyethylene glycol 1000 succincte (tpgs) have been widely used in this field [10, 11, 26, 69]. In order to reduce the amount of solidifying excipients required for transformation of sedds into solid dosage forms, a gelled sedds has been developed . Colloidal silicon dioxide (aerosil 200) was selected as a gelling agent for the oil - based systems, which served the dual purpose of reducing the amount of required solidifying excipients and aiding in slowing down of the drug release . After administration of capsules containing conventional liquid se formulations, emulsion droplets form and subsequently disperse in the gi tract to reach sites of absorption . However, if irreversible phase separation of the emulsion occurs, an improvement of drug absorption cannot be expected . For handling this problem, sodium dodecyl sulfate was added into the se formulation .with the similar purpose, the supersaturatable sedds was designed, using a small quantity of hpmc (or other polymers) in the formulation to prevent precipitation of the drug by generating and maintaining a supersaturated state in vivo . This system contains a reduced amount of a surfactant, thereby minimizing gi side effects . For handling this problem, the supersaturatable sedds was designed, using a small quantity of hpmc (or other polymers) in the formulation to prevent precipitation of the drug by generating and maintaining a supersaturated state in vivo . This system contains a reduced amount of a surfactant, thereby minimizing gi side effects . These excipients have the potential to increase the absorption of poorly water - soluble drugs relative to previously used peg solid dispersions and may also be filled directly into hard gelatin capsules in the molten state, thus obviating the former requirement for milling and blending before filling . Se excipients like gelucire1 44/14, gelucire150/02, labrasol1, transcutol1, and tocopheryl polyethylene glycol 1000 succincte (tpgs) have been widely used in this field [10, 11, 26, 69]. Capsule filling is the simplest and the most common technology for the encapsulation of liquid, semisolid, or solid se formulations for the oral route . The advantages of capsule filling are simplicity of manufacturing, suitability for highly potent low - dose drugs, and high drug loading (up to 50% w / w) potential . For liquid formulations, it involves a two - step process: filling the formulation into the capsules followed by sealing of the body and cap of the capsule, by banding or microspray sealing . Besides liquid sedds filling, the solid - sedds obtained by various techniques described above like spray drying, freeze drying, and so forth can be filled in the capsules . After administration of capsules containing conventional liquid se formulations or the solid - se formulations, emulsion / nanoemulsion / microemulsion droplets form and subsequently disperse in the gi tract to reach sites of absorption . Combinations of lipids and surfactants have presented great potential of preparing se tablets that have been widely researched . First, the self - nanoemulsion system containing the ubiquinone was prepared; this nanoemulsion was absorbed on granular materials and then compressed to form tablets . The resultant se tablets consistently maintained a higher active ingredient concentration in blood plasma over the same time frame compared with a nonemulsifying tablet . The newest advance in the research field of se tablet is the se osmotic pump tablet, in which the elementary osmotic pump system was chosen as the carrier of ses . This system has outstanding features such as stable plasma concentrations and controllable drug release rate, allowing a bioavailability of 156.78% relative to commercial carvedilol tablets . Pellets are multiple unit dosage form which possess many advantages over conventional solid dosage forms, such as flexibility in manufacturing, reduction of intrasubject and intersubject variability of plasma profiles, and minimizing gi irritation without lowering drug bioavailability . Thus, it is very interesting to combine the advantages of pellets with those of sedds by se pellets . Se controlled - release pellets were prepared by incorporating drugs into ses that enhanced their rate of release and then by coating the pellets with a water - insoluble polymer which reduced the rate of drug release . The combinations of coating and ses could control in vitro drug release and provide a range of release rates . In some investigations, solid self - emulsifying drug delivery systems (solid - sedds) were prepared by means of a wet granulation process in a lab - scale high shear mixer in order to improve the dissolution rate of a poorly water - soluble drug . The conventional liquid granulation binder was replaced with an oil - in - water microemulsion, loaded with the drug [34, 42]. Self - emulsifying system can be formulated as a solid dosage form by using minimum amounts of solidifying excipients . Patil and paradkar investigated loading ses into the microchannels of porous polystyrene beads (ppb) using the solvent evaporation method . It is inert and stable over a wide range of ph, temperature and humidity . Ppb was found to be potential carriers for solidification of ses, with sufficiently high ses to ppb ratios required to obtain solid form . Bead size and pore architecture of ppb were found to affect the loading efficiency and in vitro drug release from ses - loaded ppb . In another study, floating alginate beads containing sedds of tetrahydrocurcumin were developed to increase drug solubility and prolong gastric residence time . Use of different proportions of sodium alginate, calcium chloride, and water soluble pore former (polyvinyl alcohol - polyethylene glycol copolymer) in bead formulations was found to have different effects on the floating abilities and in vitro drug release rate . Solid se sustained - release microspheres were prepared by using the quasi - emulsion - solvent - diffusion method of the spherical crystallization technique . Zedoary turmeric oil (zto) exhibited potent pharmacological actions . With zto as the oil phase, zto release behaviour was controlled by the ratio of hydroxypropyl methylcellulose acetate succinate to aerosil 200 in the formulation . The plasma concentration - time profiles achieved after oral administration of such microspheres to rabbits showed bioavailability of 135.6% with respect to the conventional liquid sedds . Solvent injection is one of these techniques . In this method, the lipid, surfactant, and drugs this approach yielded nanoparticles (about 100 nm) with a high drug loading efficiency of 74% . The mixture of polylactide - co - glycolide (plga) and o - carboxymethyl - chitosan (o - cmc) had a se effect, with no need to add another surfactant stabilizer . Eventually the 5-fu and plasmid encapsulation efficiencies were found to have 94.5% and 95.7%, respectively, and the 5-fu release activity from the nanoparticles was found to have sustained for three weeks . Trickler et al . Developed a novel nanoparticle drug delivery system consisting of chitosan and glyceryl monooleate for the delivery of paclitaxel . These chitosan / gmo nanoparticles, with bioadhesive properties and increased cellular association, were prepared by multiple emulsion (o / w / o) solvent evaporation methods . The se property enhanced the solubility of paclitaxel and provided a foundation for chitosan aggregation, meanwhile causing near 100% loading and entrapment efficiencies of paclitaxel . These advantages allow the use of lower doses of paclitaxel to achieve an efficacious therapeutic window, thus minimizing the adverse side effects associated with chemotherapeutics like paclitaxel . Self - emulsifying phospholipid suspension (seps) consisting of high amount of phospholipids has the ability to keep the drug in solubilized form in vivo, which is essential for bioavailability enhancement . These require relatively low amount of surfactant / cosurfactant and thus posing less health problems . Some investigators proved that s - sedds could increase not only gi adsorption but also rectal / vaginal adsorption . The drugs, which do not easily achieve therapeutic plasma concentrations by oral route, may obtain satisfactory therapeutic levels for chronic hepatic diseases by either vaginal or rectal se suppositories . Self - microemulsifying suppositories of -artemether have been formulated and evaluated with the objective of faster onset of action and prolonged effect when administered by rectal route . Research into se implants has greatly enhanced the utility and application of s - sedds . As an example, 1,3-bis(2-chloroethyl)-1-nitrosourea (carmustine) is a chemotherapeutic agent used to treat malignant brain tumors . However, its effectiveness was affected by its short half - life . In order to enhance its stability compared with that released from poly(d, l - lactide - co - glycolide) (plga) wafer implants, ses was formulated with tributyrin, cremophor rh 40 (polyoxyl 40 hydrogenated castor oil), and labrafil 1944 (polyglycolyzed glyceride). Then the self - emulsified carmustine was fabricated into wafers with flat and smooth surface by compression molding . Ultimately, ses increased in vitro half - life of carmustine up to 130 min . In vitro release of carmustine from se plga wafers such wafers had higher in vitro antitumor activity and were less susceptible to hydrolysis than those wafers devoid of ses . The primary means of self - emulsification assessment is visual evaluation . To avoid any subjective variations, the% transparency of the resulting micro / nanoemulsion obtained on dilution / reconstitution of the self - emulsifying formulations the globule size of the emulsion is a crucial factor in self - emulsification performance because it determines the rate and extent of drug release as well as absorption [54, 88, 89]. It has been reported that the particle size distribution is one of the most important characteristics of the in vivo fate of drug emulsion . The globule size of the reconstituted formulations is most commonly measured using malvern zeta sizer based on the principle of dynamic light scattering (dls). Robustness to dilution is important for sedds / snedds to ensure that the emulsion / nanoemulsion formed have similar properties at different dilutions to achieve uniform drug release profile and to ensure that the drug will not get precipitated at higher dilutions in vivo which may significantly retard the absorption of the drug from the formulation [49, 90]. The seddss should be evaluated by diluting them at different dilutions and investigating their effect on the properties of the formed emulsion / nanoemulsion . The charge of the oil droplets of sedds is a property that should be assessed . Generally, the increase in electrostatic repulsive forces between the nanoemulsion droplets prevents the coalescence of nanoemulsion droplets . On the contrary, the zeta potential of the reconstituted sedds is commonly measured using malvern zeta sizer nano based on the electrophoresis and electrical conductivity of the formed nanoemulsion . The ph of the aqueous phase has considerable influence on the phase behaviour of the spontaneously emulsifying systems [33, 84]. In view of this, the effect of the ph of the aqueous phase on the resultant nanoemulsion should also be investigated . Self - emulsification has been shown to be specific to the temperature at which self - emulsification occurs [33, 52]. The viscosity of the liquid sedds is useful to assess its ability to be filled in the hard or soft gelatin capsules . If the system has very low viscosity, it may enhance the probability of leakage from the capsule and the system with too high viscosity may create problem in pourability . This test can be used to determine the stability of the sedds after emulsion formation . For this, the samples diluted with distilled water are centrifuged at specified rpm for specified time and then examined for the phase separation [92, 93]. The characterization of self - emulsifying drug delivery system can be made utilizing dye solubilization . This test is used to identify the nature of the formed nanoemulsion and its continuous phase . For this, the water - soluble dye is sprinkled onto the surface of the prepared nanoemulsion . By observing the dispersion of dye or the clump formation, when the temperature is higher than the cloud point, an irreversible phase separation will occur and the cloudiness of the preparation would have a bad effect on drug absorption, because of the dehydration of its ingredients . Hence, the cloud point for snedds should be above 37c, which will avoid phase separation occurring in the gastrointestinal tract [22, 94, 95]. Advancement of the technologies and design and development of new chemical moieties having targeting potential is leading to emergence of new drug molecules having therapeutic effect but unfavourable physicochemical properties for their drug absorption in the body . This is becoming the greatest challenge to the formulation scientists to efficiently deliver such drug molecules mostly exhibiting poor aqueous solubility . Lipidic formulations are promising approach for various categories of drug molecules having challenging drug properties . Among the various lipid formulations, the self - emulsifying delivery systems offer additional advantages of higher stability, suitability for hydrolytically susceptible drugs, high drug loading capacity, potential for oral drug delivery (solid - sedds), ease of manufacture and scale - up, and so forth, if suitably formulated with proper selection of excipients . Seddss are mostly investigated for the bcs class ii drugs having low aqueous solubility for their bioavailability enhancement and have shown promising success . They have the potential to solve the problems associated with drugs of all other classes of bcs also as summarized in table 4 . Some studies have already been performed with positive results . More investigations are needed to get better insight in the field . The challenges associated with the formulation of self - emulsifying system include the selection of right excipients with consideration of their solvent capacity, miscibility, chemical stability, capsule compatibility, self - dispersibility, regulatory issues, and so forth . The major excipients required for their formulation are the oil, surfactant, and the cosurfactant for liquid self - emulsifying systems . The criteria for the selection of the combination of excipients for sedds formulations should include their solubilising capacity for the required dose of drug, ability to self - emulsify the system when in contact with the gastric fluid (by use of phase diagram), their regulatory approval state for oral use with consideration of their permitted concentration, and so forth . The material used for transforming liquid sedds to solid forms should be inert, compatible and should not affect the emulsifying properties and the release profile of the drug . Various authors have reported insignificant effect on the emulsification properties of solid - sedds prepared by spray drying, free drying (lyophilisation), or the adsorption and extrusion technique with respect to the corresponding liquid sedds [34, 36, 45, 65]. Spray drying may be preferred because of their capability to produce smooth surfaced, well separated spherical particles at a rapid rate . But it may not be suitable for thermolabile drugs where lyophilization technique may be beneficial . Another challenge associated with the sedds is that their in vivo assessment is difficult in small animals owing to the small volume of gastric fluid in comparison to the humans which may not be sufficient for proper self - emulsification . One can foresee a good scope for the growth of the self - emulsifying drug delivery systems in near future, provided some means are developed for the estimation of their in vivo performance . There is a need for the agents which have even better self - emulsifying properties at lower concentrations to minimize any possibility of undesired effects like gastric irritation which may be associated with this system of drug delivery due to comparatively higher amount of surfactant and co - surfactant used in their formulation . A lot of investigations have been done in the field, yet there is a need for the more predictive in vitro models for predicting the changes involving the drug in sedds in the gut, so that the fate of the drug in vivo can be more reliably monitored.
Pre - implantological sinus floor augmentation serves to restore bony mass in the maxilla of partially or totally edentulous patients . Although the complication rate is low, there are intraoperative risks, such as sinus membrane perforation and bleeding, and postoperative risks of wound infection and sinusitis, graft or barrier membrane exposure, graft infection (warranting its removal), cyst formation, and flap dehiscence [215]. The panoramic x - ray can provide a sufficient view of the residual crest height under the sinus floor . For anatomical features such as the anterior or posterior wall and the septa of the sinus, the palatal curvature, and the pterygoid process, it is important to consider that panoramic image quality is highly dependent on the radiologist s skill and is a 2-dimensional image of a 3-dimensional volume with the superimposition of anatomical structures . Fortin demonstrated that the use of a panoramic radiological exam for oral implant planning in severely resorbed maxillae overestimates the need for a sinus augmentation procedure compared with the use of both 3-dimensional planning software and strategic implant placement when there is little remaining bone volume . Furthermore, this technology allows the surgeon to take advantage of the septa and palatal curvature, an option that is not routinely described in conventional procedures . To overcome the drawbacks of sinus lifting, some authors have suggested using alternative anatomic features to place the implant, such as the pterygoid process or tangential to the palatal curvature in the area of the first or second molar [1720]. For the non - grafted maxilla, krekmanov placed the implants into the pterygoid plate, palatally tilted, close to and parallel with the posterior sinus wall or close to and parallel with the anterior sinus wall . The most posterior implant was tilted distally, approximately 30 to 35 degrees . However, some authors mentioned severe complications after inserting implants into the pterygoid process . Reychler and olszewski reported a unique intracerebral penetration of a pterygoid implant inserted into the pterygoid region . An episode of acute left maxillary sinusitis, chronic fatigue, and severe headaches occurred shortly after insertion of the fixed denture . Cerebral magnetic resonance imaging (mri) demonstrated the penetration of a pterygoid implant into the middle cranial fossa . To avoid the sinus atrium and improve posterior prosthetic support in the resorbed posterior maxilla fortin was the first to demonstrate that maxillary sinus septa could be a location of interest to place implants, avoiding the sinus lift technique in a severely resorbed maxilla, in a clinical case series study . In completely edentulous patients, inserting implants into the sinus septa does not exclude the need for sinus grafting, but in partially edentulous patients, this minimally invasive technique is an alternative to subantral augmentation . Maxillary sinus septa were first described by underwood in 1910 as the walls of cortical bone within the maxillary sinus . The shape has been described as an inverted gothic arch, arising from the inferior or lateral walls of the sinus, which may divide the sinus into 2 or more cavities . Various studies were conducted on height, location, prevalence, and morphology of the maxillary sinus septa, in adult patients, using diverse medical imaging techniques such as cone beam computed tomography (cbct) [2634]. The measurements were performed in 2 dimensions (2d) in axial, sagittal, or panoramic images . The angle between the septum and the median palatine suture was also evaluated by several authors . Unlike traditional 2d radiography, cbct avoids structural superimposition and image enlargement and distortion, thus allowing precise 3-dimensional (3d) visualization and measurement of dental and maxillofacial structures, at a lower radiation dose than a multislice computed tomography (ct). To the best of our knowledge, there are still no 3d experimental studies in the literature that evaluated the thickness of the maxillary septa and the inclination of the maxillary septum plane toward the horizontal maxillary plane . The aim of this investigation was therefore to evaluate, in 3d, the maxillary sinus septa as a potential site for implant placement . Our second purpose was to compare the anatomy of the maxillary sinus septa in dentate and edentate patients to determine if immediate implant placement in maxillary sinus septa is more favorable than delayed implant placement . We selected 200 patients with maxillary sinus septa from a 3000 patient database from the department of oral and maxillofacial surgery at cliniques universitaires saint luc in bruxelles, belgium . The patients were divided into 2 groups: 100 dentate patients (group 1) and 100 edentate patients (group 2). In the first group, patients with the septum located in a dentated region were chosen . In the second group, we did not have access to information related to sex and we were only able to obtain information related to age for the dentate group . The age range of the population was from 18 to 86 years, with a mean age of 46.5318.67 years . The study was retrospective, and the indication for cbct was not considered as part of this study . Cbct images with inadequate information or that showed signs of a previous maxillary sinus surgery were excluded . The study received approval from the comit dthique hospitalo - facultaire of the universit catholique de louvain in brussels, belgium (2014/13mar/104). The cone beam ct (icat) protocol specified 120 kvp, 18 mas, 0.3 mm voxel size, 21 cm height, and a 16-cm diameter field of view . The images were examined for the presence of antral septa first on the axial slices and then on the reconstructed sagittal and coronal slices . We created maxillary sinus septa 3d reconstructions using maxilim software (medicim, mechelen, belgium). Three 3d reconstructions were prepared for each septum: 1) at the level of septum insertion on the lateral wall, 2) at the middle of the septum (green section in the middle), and 3) at the level of septum insertion on the medial wall (figure 1). 3d reconstructions were performed for each maxillary sinus and for each maxillary sinus septa when there were 2 septa present in the same maxillary sinus . Twenty - one anatomic landmarks (table 1) were identified by 1 observer twice, with a 1-week interval between identifications, on 3d reconstructions of the maxillary sinus septa to measure the septum angle, height, thickness, and length of the maxillary sinus septa (table 2). The observer was not a trained radiologist, but he was a clinically experienced oral surgeon using cbct as part of his clinical routine . For each maxillary sinus septum, we also assessed localization (anterior, middle, or posterior third), orientation (sagittal, transverse, or oblique [buccopalatal]) and if it was complete or partial (incomplete septa on the sinus floor and on the medial or lateral sinus wall). We considered a septa complete when the bony crest was inserted both on the lateral wall and on the medial wall of the maxillary sinus . The orientation was defined according to the angle between the septum plane and the median palatine suture and separated into 3 categories: 1) transverse if at 90 with the median palatal suture, 2) oblique if less or more than 90, and 3) sagittal if parallel to the medial palatal suture . The intraclass correlation coefficient (icc) showed that there was no statistically significant difference between the 2 intraobserver measurements (p>0.05). Ninety - eight percent of the dentate patients and 96% of the edentate patients presented with complete septa . Two percent of the dentate patients and 4% of the edentate patients presented with incomplete septa . The analysis of the anatomic location of the septa within the maxillary sinus was performed only in dentate patients and revealed that 55.4% were located in the posterior region, followed by the middle region (33.7%) and the anterior region (10.9%). For dentate patients, the spatial orientation of the septum was transverse in 18.8% of patients and oblique in 81.2% of patients . The range and mean values for septum angle, length, height, and thickness are shown in table 3 . Middle height was the only distance that was different between edentate and dentate patients (p=0.0095, edentate mean <dentate mean). There were no statistically significant differences among the 2 groups, except for the middle height . Qian observed a significantly (p<.05) greater occurrence rate of 57.4% in the edentulous group compared with the rate of 39.7% observed in the dentulous group . The results of our study concerning the morphology of the septa are different from those of other studies . Confirmed that 40% of patients have bony septa that can partially separate the maxillary sinus . In our study, 98% of the dentate patients and 96% of the edentate patients presented with complete septa . Excluded bony septa less than 4 mm in height or width . As for the location of the septa, a greater number were found in the posterior regions (55.4%), but several studies observed them in the anterior and in the middle regions . Differences in localization may be because we did not exclude secondary septa such as bony crests on the maxillary sinus floor, also known as secondary septa, which can be considered a result of tooth loss and atrophy . To the best of our knowledge, there are no 3d studies that evaluated the inclination of the septum plane on the horizontal maxillary plane . The results of this study indicate that the angle formed by the septum plane and the horizontal maxillary plane is favorable for implant insertion (86.03 for dentate patients and 89.38 for edentate patients). According to malo, the implant can be placed from a vertical position in the canine / first premolar region to a tilted implant in the second premolar / first molar region, following the anterior sinus wall up to 45 degrees of inclination . The reproducibility of the 3 landmarks that defined the maxillary horizontal plane was not evaluated . Previous studies reported different heights for the septa, ranging from 1 to 42 mm . The mean septum heights in our study groups were lower than those found in other studies . In comparison with other studies, cbct is an alternative to conventional multislice ct, with a low radiation dose (4 to 10 times lower, depending on the volume scanned). For both study groups, we are the first to use the technique of 3d imaging to assess the thickness of the septa, along its lateral - medial dimension . Our results indicate that the lateral and medial aspects of the septa are the most likely to increase available alveolar bone height for implant placement . Further studies should focus on improving the accuracy of the 3d evaluation method and on the potential for using sinus septa to clinically evaluate virtual implant placement into the maxillary sinus septa . . Extensive evaluation of sinus septa using 3d imaging showed that the sinus septum can increase the available bony height for implant placement.
Accumulation of smooth muscle cells (smcs) in the tunica intima plays a crucial role in the pathogenesis of vascular lesions, including atherosclerosis and restenosis following angioplasty or stenting procedures (1, 2). In the classical pathogenetic hypothesis of atherosclerosis, following injury growth factors and proteolytic agents induce smc proliferation and migration from the tunica media into the intima (3). During this process, phenotype, characterized from reduced expression of contractile proteins and enhanced response to growth and chemotactic factors (4). In atherosclerosis, smc accumulation in the fibrous cap is monoclonal or oligoclonal (5, 6), implying that only a small number of medial smcs proliferate in response to injury . This finding suggests the existence of a resident arterial stem cell subpopulation contributing to arterial healing in response to injury . Studies in human transplantation arteriopathy and primary atherosclerotic lesions showed that recruited bone marrow or host - derived circulating precursors are present in the lesions (68). Successively, other studies refused this hypothesis (9, 10), suggesting that smcs in vascular lesions are mainly derived from the host parietal response . In parallel, the presence of cells expressing stem cell antigens have been identified in the normal arterial wall (11, 12), supporting a potential role of adult vascular wall - resident stem cells (vrscs) in vascular pathobiology . In this review, the characteristics and the potential contribution of vrscs to the development of great vessel lesions are discussed . Classically, smcs within the normal tunica media of the adult vascular wall are heterogeneous, and a contractile and synthetic phenotype identified (13, 14). Smcs displaying a synthetic phenotype can be identified more frequently in pathologic arteries and have been characterized in vitro (fig . 1). Synthetic smcs obtained from intimal aortic tissue fifteen days after balloon injury displayed a monolayered epithelioid phenotype, with a cobblestone morphology, markedly different from the hill - and - valley moreover, neointimal cells express low amount of myocitic markers and differences are maintained in clonal expansion (1416). It is worth of noting contractile and synthetic phenotypes are not permanent and can partially revert after stimulation with growth factors and extracellular matrix molecules (1719). Phenotypically - regulated activation of proteins and receptors regulates differences in terms of proliferation and survival, being proliferating aortic neointimal vsmcs more sensitive to apoptosis (20). Likely linked to a phenotype - regulated different nf-b activity (21, 22). Phenotype - dependent expression of in - tegrins in vascular smc regulates morphology, motility and gene expression in collagen matrix (fig . 1; 18). A hypothesis to explain smc heterogeneity in adult vessels is a different embryologic origin during vasculogenesis (23). In the chick embryo aorta, spindle - shaped and epithelioid phenotypes responding differently to tgf- were isolated from distinct mesoderm and neural crest - derived regions (24). It is likely that the capacity of a different response to damage can be retained from smcs during the adult life . Mesenchymal smooth muscle progenitors have been identified in the bone marrow, in the blood as circulating progenitors and in extravascular sites (2527). Identification of these progenitor cells was mainly based on the finding of stem antigens shared from a smc subpopulation in the normal arterial wall and/or in vascular lesions . Recent studies identified and characterised a small population of resident smcs in the wall of great vessels of healthy adult mice expressing sca1 and low amounts of c - kit and cd34, absent in the adventitia (28). These putative vrscs differ from bone marrow - derived smooth muscle progenitors or form those isolated from skeletal muscle, since they lack the ability to differentiate into lymphoid or myeloid lineages (29). A clonal subpopulation of vascular cells from the bovine aortic tunica media was phenotypically similar (cd29, cd44, cd14, cd45) to bone marrow - derived mesenchymal stem cells (30). Progenitor cells named mesoangioblasts isolated from explants of murine dorsal aorta display differentiative potential into various mesenchymal cell types other than myocitic cells in vitro and express both myogenic and endothelial markers (31, 32). Immunohistochemical investigation revealed c - kit and cd133 cells in human atherosclerotic plaques and restenosis lesions and in rat aortic post - injury neointima, whereas smcs in the tunica media of primary atherosclerotic plaques and normal arteries were c - kit- (11, 33). Progenitor cells expressing sca-1, cd34, kdr and c - kit were detected within human plaques and the adventitia (11). Most of the knowledge concerning stem phenotype derives from studies of animal models of vascular injury . Vegfr-1 or flt-1 and c - kit smcs are extremely rare in normal rat aorta, whereas their number dramatically increases fifteen days after injury (fig . 2), with many of intimal cells co - expressing both -smooth actin and flt-1 or c - kit (12). After sixty days, after complete re - endothelialization stem marker - expressing intimal cells almost disappeared and -smooth actin expression restored (12). Double immunostaining experiments revealed that flt-1 medial are also positive for anti - bromodeoxyuridine (fig . 3) or apoptotic tunel staining, confirming the preferential involvement of flt-1 cells in proliferative and apoptotic behaviour (22, 34, 35). Similarly, many intimal cells of mice carotid arteries express hedgehog - induced notch 1 early after injury (36). Although recent studies supported the hypothesis that the majority of smcs in vascular lesions originates from the vessel wall, an open question remains the layer of origin of vrscs . After early reports documenting that activation of adventitial fibroblasts precedes post - damage experimental neointimal proliferation (37), increasing evidence accumulated supporting the involvement of adventitial vascular progenitor cells in the development of arteriosclerosis, including transplant arteriosclerosis, angioplasty - induced restenosis, vein graft atherosclerosis, and spontaneous atherosclerosis . The adventitia of the arterial wall contains progenitor cells, which can differentiate into vascular smcs . In apoe - deficient mice these progenitor cells were able to migrate from the adventitia into the intima, where they accumulate to contribute to athero - sclerotic lesions of vein grafts (38). (2004) showed documented aortic adventitial cells in apoe - deficient mice expressing sca-1 and c - kit stem cell markers; moreover, ex vivo pdgfb - stimulated adventitial stem cell - expressing cells displayed a myocytic phenotype with the expression multiple smooth muscle markers (39). Lineage tracking with bone marrow transplantation from rosa 26 lacz gene - expressing transgenic mice documented that adventitia - derived smooth muscle precursors migrate to the neointima in a vein graft atherosclerosis model, with no evidence of a bone marrow origin (39). The same authors suggested that the extent of injury is crucial to regulate mobilization of adventitial progenitor cell and their contribution to vascular lesions . Several issues remain unresolved as to the physiologic relevance of adventitial progenitor cells, and if these cells are also documented in human lesions . Recently, an adipose - derived stem population has been well characterized (4045). Adipose derived stem cells are capable to regenerate de novo mesenchymal tissue and are widely used for their pluripotential regenerative potential (4652). In most of these studies, vascular transdifferentiation is reported as a crucial event to explain clinical efficacy of adipose - derived stem cells to ameliorate grafting and tissue regeneration . It is interesting to note that, differently from adipose - derived stem cells, bone marrow - derived circulating progenitor cells do not transdifferentiate into adipocytes in vivo and play little, if any, role in expanding the number of resident adipocytes during tissue growth (53). These findings strongly support that de novo vessel formation and/or vascular remodelling involve transdifferentiation of adventitial adipose - derived stem cells . Conversely, in long - term cultures adipose tissue - derived perivascular cells retained myogenicity and mesenchymal stem cell markers expression, similar to native, non - cultured perivascular cells (53). The vasculogenesis - promoting potential of adventitial adipose - derived stem cells depends on interaction with resident endothelial cells and involving contact and bi - directional interaction, resulting in modulated secretion of cytokines and extracellular matrix molecules, so leading to vascular remodeling (54). Cellular communication occurring between adventitial adipose - derived stem cells and endothelial precursor cells and triggered by cell - cell contact is partially mediated by secreted vegf, but this effect is not observed by using huvecs, suggesting that adipose - derived stem cells are unique in this sense (55). Being adventitial precursors a potential source of smcs, the modulation of the contribution of these progenitor cells is a potential tool to counteract pathological vascular remodeling by cellular, genetic, and tissue engineering approaches . Blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of non - vascular tissues as other related adult stem cells (56). Recently, resident pericyte - like cells from the adventitia have been hypothesized to contribute to restenosis . Rare ng2, pdgfr and cd146-expressing cells identified in the adventitia of uninjured mouse femoral arteries in response to injury increased in number and expressed a smc - like phenotype (31). Bone marrow transplantation and ex vivo artery culture strongly supported that pericyte - like progenitor cells are not bone marrow - derived, but originated from the adventitia after proliferation of resident perivascular pericyte - like cells (32). Cells with progenitor pericyte - like properties have been also isolated from the tunica media of adult rat aortas and are capable to generate spheroidal colonies in suspension and, when serum - cultured, retain cd34 negativity and express de novo smc markers (32). The relationship between this population of resident adventitial pericyte - like cells, reparative myofibroblasts, normal media smcs and other progenitor populations during pathological vascular remodelling is still matter of debate and need further investigation . The discovery of the existence of vrscs has challenged the classical pathogenetic model of tunica media as unique source of smcs contributing to vascular lesions (1, 2). Atherosclerosis is a chronic inflammatory disease, in which risk factors result in dysfunction and damage to the arterial endothelium and slow progression, characterized from intimal accumulation of smcs in a fibromuscular cap covering a cholesterol - rich necrotic core (1). Both smcs and monocyte - macrophages proliferate, accumulate and undergo apoptosis into the neointima (1, 2, 34, 35). In restenotic lesions, after acute injury, rapid proliferation and subsequent accumulation of smcs from the tunica media into the intima results in rapid lumen occlusion . In these lesions, smc progenitors have been identified by the expression of bone marrow or progenitor cell markers in vivo . Green florescent bone marrow transplantation in mice revealed that bone marrow - derived cells contribute up to 60% of smcs in the post - injury neointima (7). Other studies report that donor - derived cells represent up to 10% of smcs within atherosclerotic lesions in humans receiving sex - mismatched bone marrow transplantation (8). The major limit to these investigations was the method to estimate stem marker expression in lesional smcs and accuracy of co - localisation of two markers, being confocal microscopy rarely performed . When accurate techniques were applied, even in transplant arteriopathy studies, neointimal smcs appeared to originate mainly from the recipient aortic wall (6, 9, 10). Moreover, other studies report that expression of stem markers is present only in early post - injury lesions, questioning about the transient nature of stem phenotype and the survival of these cells (22). Moreover, many studies employed -smooth muscle actin as myocitic marker, but the latter is generally down - regulated in smcs of vascular lesions (13), but strongly expressed in myofibroblast reparative cells (16). Consequently, those recent studies strongly support the hypothesis that the majority of smcs in advanced atherosclerotic lesions and healing after plaque rupture originates from the vessel wall . Nevertheless, enhanced adventitial - derived progenitor cell recruitment and increased neointima formation has been seen after growth factor stimulation in animal models of vascular injury (12, 37, 38), so favoring vascular lesion progression . More recently, it has been reported that smooth muscle progenitor cell enrichment reduces the progression of early but not advanced atherosclerotic lesions plaques in mouse aortas, suggesting a favourable effect on plaque stabilization (57). These findings support a beneficial role of smooth muscle progenitor cells in suppressing atherosclerosis and its clinical sequelae, but still their parietal or extraparietal origin remain under investigation (58). More accurate studies demonstrating whether vrscs either protect or promote vessel from pathological remodelling are needed, in order to promote cell - based or pharmacological approaches aimed to prevent vascular diseases (59). Accumulation of vascular smooth muscle cells in the tunica intima plays a major role in the pathogenesis of atherosclerosis . Aging represents a risk factor for the development of vascular lesions (6063) and the prevalence of atherosclerotic lesions in aged subject must be considered in therapeutic and experimental approaches (19, 64). Beside proliferation and differentiation, intrinsic aging enhances stem antigen expression in aortic smcs (65) and, in parallel, susceptibility to atherosclerosis (60). Atherosclerotic lesions and also macroscopically normal human and rat aortas show an increased number of vegfr-1, c - kit and sortilin cells in the intimal thickening (65, 66). Both circulating and resident progenitor cells have been evocated to contribute to the response of the adult arterial wall to damage during atherogenetic process and aging (58). Inflammatory cell recruitment induces vegfr-1-mediated physiological and pathological angiogenesis that favors the increase of vessel lumen increase and its stabilization and counteracts pathological angiogenesis stimulated from plgf - mutated variants that not bind vegfr-1 (67, 68). These findings suggest that with aging stem cells with a synthetic vegfr-1 myocitic phenotype prevail and contribute to aortic myointimal thickening and to vascular angiogenetic / arteriogenetic and healing processes (69, 70). If this population derives from perpetual proliferation of vrscs or phenotypic conversion of previously differentiated smcs is still object of debate . Smcs within the normal tunica media of the adult vascular wall are heterogeneous, and prevalence of a synthetic starting from smc heterogeneity, a considerable accumulating evidence suggests the presence of a small population of vrscs in different layers of the normal adult arterial wall, and its potential contribute to the homeostasis of post - natal arterial wall but also to pathological remodelling . Progenitor cell mobilisation may be tracked using genetic markers or co - localisation of stem and myocitic markers, but still variability of results is encountered according to the different experimental procedures and techniques . Further accurate studies are needed to define the positive or negative role of vrscs in arterial pathobiology and to identify if the selective control of vrsc intraparietal mobilization may represent an attractive therapeutic opportunity.
Subjects in the present study are part of a longitudinal study of the etiology of type 2 diabetes among the gila river indian community in arizona, where most of the residents are pima indians or of the closely related tohono o'odham tribe . Diabetes status was determined by an oral glucose tolerance test according to the criteria of the world health organization (10). The initial genetic study was conducted in a population - based sample of full - heritage pima indians (n = 3,501), where 1,561 subjects had type 2 diabetes (37% male, age at the last exam 49 14 years, and bmi 39 8 kg / m) and 1,940 subjects were nondiabetic (46% male, age at the last exam 31 15 years, and bmi 36 8 kg / m). Independent replication was assessed in 3,723 subjects from the same longitudinal study who were of mixed native american heritage (reported heritage, on average, was one - half pima and three - quarters native american). The replication sample had 750 subjects with type 2 diabetes (41% male, age at the last exam 42 14 years, and bmi 38 9 kg / m) and 2,973 nondiabetic subjects (47% male, age at the last exam 24 11 years, and bmi 34 8 additional replication was assessed in a case - control sample from the old order amish (139 diabetic subjects and 347 subjects with normal glucose tolerance) as previously described (11). Among the full - heritage pima indians, 415 subjects (58% male, age 27 6 years, and bmi 34 8 kg / m) had undergone detailed metabolic testing for risk factors that predict type 2 diabetes . These individuals were determined to be nondiabetic, and acute insulin response was only analyzed in subjects who had normal glucose tolerance . Glucose tolerance was determined by a 75-g oral glucose tolerance test (ogtt) with measurements of fasting and 30, 60, 120, and 180-min plasma glucose and insulin concentrations (12). The acute insulin response to intravenous glucose was measured on a separate day from the ogtt . Blood samples were collected prior to a 25-g glucose intravenous bolus infusion and at 3, 4, 5, 6, 8, and 10 min after infusion . The acute insulin response was calculated as the mean increment in plasma insulin concentrations from 35 min (13). Insulin sensitivity was assessed using the hyperinsulinemic - euglycemic clamp technique as previously described (12,13). Body composition was estimated by underwater weighing until january 1996 and by dual - energy x - ray absorptiometry (dpx-1; lunar radiation) thereafter (14). Dna from 24 pima indians (12 nondiabetic and aged> 45 years; 12 diabetic with onset age <25 years) was sequenced using a big dye terminator (applied biosystems) on an automated dna capillary sequencer (model 3730; applied biosystems). Genotyping was done using the snplex genotyping system 48-plex (applied biosystems) on an automated dna capillary sequencer (model 3730; applied biosystems) for the native american samples and taqman genotyping assays (applied biosystems) for the old order amish samples . In the pima study, statistical analyses were performed using the software of the sas institute (cary, nc). The general association of genotypes with type 2 diabetes was assessed by logistic regression analysis and was adjusted for covariates (age, sex, and birth year). The model was fit with a generalized estimating equation technique to account for correlation among siblings . Genotype was analyzed as a numeric variable representing the number (0, 1, 2) of copies of a given allele . The association of quantitative traits with genotypes was analyzed by linear regression using the generalized estimating equation procedure to account for correlation among siblings . P values were adjusted for potential confounding covariates . In the replication study, which included individuals of mixed ancestry, these estimates were derived by the method of hanis et al . (15) from 39 informative markers with large differences in allele frequency between populations (16). Linkage disequilibrium (ld) and haplotype blocks were estimated by haploview (version 3.32). For the amish study, the odds ratio (or) is derived from a logistic regression model, while the p value is based on the normal - liability threshold model implemented in solar to account for relationships among individuals . Among the full - heritage pima indians, 415 subjects (58% male, age 27 6 years, and bmi 34 8 kg / m) had undergone detailed metabolic testing for risk factors that predict type 2 diabetes . These individuals were determined to be nondiabetic, and acute insulin response was only analyzed in subjects who had normal glucose tolerance . Glucose tolerance was determined by a 75-g oral glucose tolerance test (ogtt) with measurements of fasting and 30, 60, 120, and 180-min plasma glucose and insulin concentrations (12). The acute insulin response to intravenous glucose blood samples were collected prior to a 25-g glucose intravenous bolus infusion and at 3, 4, 5, 6, 8, and 10 min after infusion . The acute insulin response was calculated as the mean increment in plasma insulin concentrations from 35 min (13). Insulin sensitivity was assessed using the hyperinsulinemic - euglycemic clamp technique as previously described (12,13). Body composition was estimated by underwater weighing until january 1996 and by dual - energy x - ray absorptiometry (dpx-1; lunar radiation) thereafter (14). Dna from 24 pima indians (12 nondiabetic and aged> 45 years; 12 diabetic with onset age <25 years) was sequenced using a big dye terminator (applied biosystems) on an automated dna capillary sequencer (model 3730; applied biosystems). Genotyping was done using the snplex genotyping system 48-plex (applied biosystems) on an automated dna capillary sequencer (model 3730; applied biosystems) for the native american samples and taqman genotyping assays (applied biosystems) for the old order amish samples . In the pima study, statistical analyses were performed using the software of the sas institute (cary, nc). The general association of genotypes with type 2 diabetes was assessed by logistic regression analysis and was adjusted for covariates (age, sex, and birth year). The model was fit with a generalized estimating equation technique to account for correlation among siblings . Genotype was analyzed as a numeric variable representing the number (0, 1, 2) of copies of a given allele . The association of quantitative traits with genotypes was analyzed by linear regression using the generalized estimating equation procedure to account for correlation among siblings . P values were adjusted for potential confounding covariates . In the replication study, which included individuals of mixed ancestry, (15) from 39 informative markers with large differences in allele frequency between populations (16). Linkage disequilibrium (ld) and haplotype blocks were estimated by haploview (version 3.32). For the amish study, the odds ratio (or) is derived from a logistic regression model, while the p value is based on the normal - liability threshold model implemented in solar to account for relationships among individuals . Sequencing of the mbl2 gene (all four exons, three introns, and 2 kb of the upstream region) in 24 pima indians identified 37 variants . Rs5030737 (arg52cys), rs1800450 (gly54asp), and rs1800451 (gly57glu)commonly referred to as a / d, a / b, and a / c, respectively (12). Three known promoter snps, rs11003125, rs7096206, and rs7095891, previously classified as 550 g> c (h / l), 221 g> c (y / x), and + 4 c> t (p / q), respectively, were also identified (3). From these 37 snps in the mbl2 gene, 12 tag snps were selected based on the tagger algorithm (haploview 3.32, using r 0.8 to indicate redundancy [supplementary fig . 1, available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1593/dc1]). To additionally analyze variation flanking mbl2 (25 kb flanking each side of the sequenced region, chromosome 10:5417014654228466), 20 database snps that serve as tag snps in the chinese hapmap (r 0.8; minor allele frequency 0.1) were genotyped in pima indians, and it was determined that seven snps could serve as tag snps (defined above) in pima samples (supplementary fig . All 19 tag snps (12 in mbl2 and 7 flanking) were genotyped in the sample of 3,501 full - heritage pima indians for association analysis with type 2 diabetes (table 1). The missense snp rs1800450 (gly54asp, designated a / b) and the promoter snp rs11003125 (designated h / l) were in high ld (d = 0.99; r = 0.71) and were associated with type 2 diabetes (p = 0.002 and 0.0007, respectively, adjusted for age, sex, birth year, and family membership; table 1). These two tag snps captured several additional snps within intron 2 and the region near mbl2 . Associations of mbl2 tag snps with type 2 diabetes in pima indians nineteen tag snps (r 0.8) were selected from 57 snps that span mbl2 (8.3 kb) and approximately 25 kb flanking each side of the gene . Allele frequency (af) is presented as frequency of the risk allele . Or is expressed as per copy of the risk allele (and thus, by definition, is> 1). Sequences flanking the two novel snps are as follows: novel: promoter, tttcatggatgggtgtgtgc[g / a]tgcatgcacgtgtctgtgtg; novel: 3utr, catgactgcacagtaatttc[g / a]tctgtttataaacattgtat . To determine whether the association with type 2 diabetes in the full - heritage pima indians could be replicated in other native americans, rs1800450 and rs11003125 were further genotyped in a nonoverlapping sample of 3,723 subjects who were predominately of mixed native american heritage . The promoter snp rs11003125 reproducibly associated with type 2 diabetes (p = 0.04, adjusted for age, sex, birth year, and heritage) (table 1). Combining the initial and replication samples (n = 7,224) provided the strongest evidence for association with type 2 diabetes for rs11003125 (p = 9.2 10) (table 1). The combined sample also showed a significant association for rs1800450 (p = 0.001) (table 1), but this significant association was largely driven by the initial full - heritage pima sample, with only a nonsignificant association in the same direction (p = 0.18) identified in the largely mixed - heritage replication group . To determine whether variants in mbl2 had a significant effect on diabetes in non native american populations, rs1800450 and rs11003125 were genotyped in an amish sample of 139 diabetic subjects and 347 subjects with normal glucose tolerance . Consistent with the native american samples, rs11003125 was associated with type 2 diabetes in the amish (or 1.51; p = 0.004, adjusted for age, sex, and family structure) (table 2), but the frequency of risk allele g was lower in the amish than in pima indians (0.45 vs. 0.77, respectively). The snp rs1800450 (gly54asp) was also associated with type 2 diabetes in the amish (or 2.38, adjusted p = 0.003) (table 2), but the frequency of risk allele glycine was comparable in the amish and pima indians (0.87 vs. 0.83). In contrast, these associations do not appear to replicate in the large caucasian diabetes genetics replication and meta - analysis (diagram) (19). Neither of these snps were directly genotyped in genome - wide association (gwa) studies from the diabetes genetics initiative (dgi) and the wellcome trust case control consortium (wtccc), which are two of the three large studies from which the meta - analysis was derived; however, a proxy, rs1838065, which we determined to have an r = 1 with rs11003125 (based on our genotyping of 90 caucasians), was not associated with type 2 diabetes in diagram (p = 0.35, table 2). Combining the negative diagram data together with the positive amish and native american data rendered the overall combined association nonsignificant (p = 0.17) (table 2). Association of promoter rs1103125 and rs1800450 (gly54asp) with type 2 diabetes in pima indian, amish, and diagram subjects a combined test was conducted by the inverse variance method . Ors are per copy of the risk allele identified in the full - heritage pima sample . Phet is the p value for the null hypothesis that the ors are the same for the combined groups . * diagram results based on rs1838065, which has an r of 1.0 with rs1103125 . Data across a larger genomic region encompassing mbl2 could also be obtained from prior gwa studies in pima indians (20), dgi (21), wtccc (22), and diagram (19). For example, a 400 kb region encompassing the 8.3 kb mbl2 (chromosome 10: 5400702754401287) yielded 101 snps that were previously genotyped in a gwa study of pima indians . Only one gwa study snp (rs1838065) was located within mbl2 (intron 2). The pairwise ld pattern of these 101 snps and their association with early - onset type 2 diabetes in pima subjects who were analyzed in this prior gwa (300 early - onset diabetes case and 334 control subjects) are shown in supplementary fig . 2a and b. gwa study snp rs1838065, which is in near - perfect ld with the promoter rs11003125 (r = 0.99), was associated with early - onset type 2 diabetes (defined as diabetes onset 25 years of age) in the gwa study (adjusted p = 0.0006), as were several nearby gwa study snps (adjusted p = 0.00070.005) (supplementary fig . 2), which were in high ld among themselves (r = 0.660.99) but in low ld with rs1838065 (r = 0.300.37). In contrast with rs1838065, these additional gwa study snps (tagged by rs920727) had only a borderline association with type 2 diabetes (defined as diabetes at any age) in the current larger study of 3,501 full - heritage pima subjects (rs920727, p = 0.05) (table 1). Gwa study data across this region in the dgi and wtccc studies and diagram are shown in supplementary fig . Overall, there were no consistent associations with type 2 diabetes across the region in any of these caucasian studies . When gwa study results of pimas were compared with other populations in the mbl2 region, rs1838065 was not significant in diagram . Snps tagged by rs17587392 in caucasians had a borderline significance with type 2 diabetes in diagram (p = 0.05) (supplementary fig . 3) but was nearly monomorphic (minor allele frequency <0.001) and therefore uninformative in full - heritage pima indians, while snp rs11003132, which had a borderline association with type 2 diabetes in wtccc (p = 0.05) (supplementary fig . 3), was not associated with type 2 diabetes in pima indians (table 1). The observation that specific mbl2 snps had replicated associations with type 2 diabetes in native americans and a small group of amish subjects, who are of european descent but were not associated with diabetes in the large diagram, is unexpected ., both the pima indians and the old order amish represent far more homogeneous populations compared with the study population of diagram . It is also possible that susceptibility genes for common diseases may have larger effects in these populations compared with others as a result of segregation of high - penetrance alleles that are rare or nonexistent in the general population, gene - gene or gene - environment interactions, or the absence of other susceptibility genes whose effects could mask other genes . To aid in validating the positive associations with type 2 diabetes, we further investigated whether these variants in mbl2 were associated with metabolic risk factors that predict type 2 diabetes among 415 nondiabetic, full - heritage pima indians . For both rs11003125 and rs1800450, the allele associated with higher risk for diabetes (g and glycine, respectively) was associated with a higher 2-h plasma glucose concentration (adjusted p = 0.0005 and 0.04, respectively) and higher 2-h plasma insulin concentration (adjusted p = 0.0008 and 0.003, respectively) during an oral glucose tolerance test (table 3). The risk allele glycine for rs1800450 was additionally associated with a lower acute insulin response to an intravenous glucose bolus infusion (adjusted p = 0.004) among subjects who had normal glucose tolerance (n = 281) (table 3). Metabolic characteristics of full - heritage nondiabetic subjects grouped by genotypes of promoter rs11003125 or rs1800450 (gly54asp) variants data are means se unless otherwise indicated . Plasma insulin concentrations (fasting, 2 h, and 30 min), rates of glucose disappearance during the low - dose insulin stimulation, and the acute insulin response were log transformed before analyses to approximate a normal distribution . The p value for percent body fat was adjusted for age, sex, and family membership . The p value for the acute insulin response was adjusted for age, sex, percent body fat, and rate of glucose disappearance during the low - dose insulin stimulation . All remaining p values (except for age) were adjusted for age, sex, percent body fat, and family membership . Previous functional studies have shown that serum mbl levels are greatly influenced by variants within the mbl2 gene . Three promoter variants (rs11003125 [h / l], rs7096206 [y / x], and rs7095891 [p / q]) and three missense variants (rs5030737 [a / d], rs1800450 [a / b], and rs1800451 [a / c]) were previously associated with mbl deficiency (3,23). Several of these variants are in high ld, so a limited number of haplotypes are present in humans . Hypa (g - g - c - gly) haplotype carriers have the highest serum concentration of mbl, typically from 1,400 to 2,500 g / l, whereas lypb (c - g - c - asp) haplotype carriers have the lowest serum concentration: 20400 g / l (3,20). Since rs7096206 (y / x) and rs7095891 (p / q) are very rare in pima indians (minor allele frequency <0.03), these high versus low serum level haplotypes can essentially be determined from a two - snp haplotype of the promoter rs11003125 (h haplotype analysis for rs11003125 and rs1800450 was performed in the combined sample of 7,224 predominately pima indians . The haplotype g - glycine (ha), carrying both the g nucleotide (designated h allele) of the promoter rs11003125 and glycine (designated a allele) of rs1800450, was associated with increased risk for type 2 diabetes in pima indians (haplotype frequency of g - glycine 0.78 in diabetic vs. 0.71 in nondiabetic; or 1.25 [95% ci 1.121.33], p = 0.0001, adjusted for age, sex, and birth year [heritage estimate in the replication group]). Because this haplotype g - glycine is highly concordant with the g (h) allele of rs11003125 (r = 0.99), it is difficult to statistically distinguish the single genotypic versus haplotypic effects . When both snps are included in a single model, there is a significant association with rs11003125 (or 1.29 [95% ci 1.081.54]; p = 0.005) conditional on the effect at rs1800450, but there is little association with rs1800450 conditional on the effect at rs1103125 (0.94 [0.771.16]; p = 0.61). It thus appears that the promoter rs11003125 is the stronger predictor of diabetes, with little additional information from rs1800450 . The prevalence of type 2 diabetes was plotted in the full - heritage pima indian (n = 3,081) (fig . 1b) groups according to the genotypes of rs11003125 and rs1800450 . Ordering the genotypic groups according to their association with serum mbl levels showed that subjects homozygous for both g and glycine alleles had a higher prevalence of diabetes than did subjects homozygous for the c allele and either homozygous or heterozygous for the aspartic acid alleles (ptrend = 8.4 10 in the combined analysis of 6,585 predominately pima subjects, adjusted for age, sex, and birth year, and in the replication group, heritage). In caucasians, eskimos, africans, south americans, and native americans, the g - glycine (ha) haplotype is associated with higher mbl serum level (3,2426). Prevalence of type 2 diabetes in the full - heritage pima indian group (a) (n = 3,081) and the replication mixed heritage group (b) (n = 3,504) according to genotypes of promoter rs11003125 and rs1800450 (gly54asp). * either homozygous or heterozygous for the asp alleles . The present study demonstrates that an allele for mbl2, which arises predominately from the promoter snp rs11003125 (g allele), predicts a higher serum level of mbl2 and is associated with increased risk for type 2 diabetes, increased 2-h plasma glucose and 2-h plasma insulin, and decreased insulin secretion in some populations . Consistent with our observations, high mbl2 levels have recently been reported to be associated with high a1c levels in the strong heart study, a longitudinal study of cardiovascular disease among native americans (26). Snp rs11003125 is in high ld with other snps that map within intron 2 and a flanking region of mbl2; therefore, the contribution of other functional snps cannot be ruled out . Nevertheless, both high ld and diabetes association were restricted to the region in and near mbl2, suggesting that evidence for association with type 2 diabetes is more likely derived from mbl2 rather than other genes in the region . Although the physiologic mechanisms underlying the association of mbl2 levels with diabetes are unknown, it has previously been shown that mbl2 plays a dual role in modifying inflammatory responses (27). Deficiency of mbl has been linked to increased risk of developing type 1 diabetes (6), insulin resistance, and obesity (9) as a result of a chronic infectious state or low - grade inflammation . Mbl2 could also affect metabolic pathways through stimulating fatty acid oxidation in skeletal muscle (28) or reducing release of tumor necrosis factor-, interleukin-1, and interleukin-6 (29). In contrast, increased mbl levels could lead to an overly activated complement system, thereby inducing inflammatory damage or interweaving a complex autoimmune process (30). Consistent with the latter effect, high mbl levels have been associated with increased risk for insulin resistance in pregnancy (31) and late - onset of rheumatoid arthritis (5). However, our study indicates that mbl2 variants are more likely to influence type 2 diabetes via an effect on insulin secretion rather than on insulin action, suggesting that inflammatory damage in pancreatic -cell function may be involved . Additional studies are needed to investigate the impact of this gene on specific type 2 diabetes related pathways and disease susceptibility in non native american groups.
Middle ear cholesteatoma is defined pathologically as the abnormal existence of keratinized squamous epithelium in the middle ear cavity, leading to bone destruction of middle ear structures and their surroundings, and is characterized by unusual hyperkeratosis and hyperproliferation . Epithelial homeostasis in normal skin is maintained by a delicate balance between proliferation and terminal differentiation rate.1) on the other hand, it has been proposed that the development of human cholesteatoma is due to altered control of cellular proliferation, which tilts the balance toward the aggressive, invasive growth of squamous epithelium into middle ear . Histopathologically, basal cell hyperplasia, appearance of suprabasal cells with new proliferation capacity are observed in cholesteatoma, resulting in disruption of epidermal homeostasis between proliferation and terminal differentiation rate.2 - 4) involucrin is precursor of human cornified envelope and can be a marker for the intermediate stage of squamous differentiation.5,6) filaggrin is a major component of keratohyalin, and is concerned with differentiating of granular and corneal layer.7,8) cytokeratins (ck) are known to be insoluble proteins that form the intermediate filaments of mammalian cells, and have been introduced as markers of cellular proliferation.9,10) the aim of this immunohistochemical study is to determine whether the hyperproliferative and hyperkeratotic characters of cholesteatoma are associated with differentiation of keratinocytes in cholesteatoma in comparison to retroauricular skin (ras), by examining the localization of marker proteins, such as involucrin, filaggrin, and cks, which is typically expressed in different steps of squamous epithelial differentiation . We obtained cholesteatoma tissue from 30 subjects during surgery as study group, and normal ras tissue from 10 subjects as control group . Immunohistochemical staining was performed to evaluate the expression of involucrin, filaggrin, ck 4, ck 10 and ck 16 . The tissues were fixed in 10% buffered neutral formalin solution and embedded in paraffin . For immunohistochemical staining, 4 m - paraffin embedded tissue sections were prepared on leica plus slide (leica biosystems richmond, inc ., the tissue sections were deparaffinized 3 times in dewax solution (leica biosystems newcastle ltd ., newcastle, uk) for 1 minute at 72, and then treated 4 times in 100% ethyl alcohol 1 minute respectively, prior to rehydration with distilled water . Antigen retrieval was performed by heating the slides for 15 minute in epitope retrieval solution 1, 2 (leica biosystems newcastle ltd ., the slides were then treated with 3% h2o2 in bond polymer refine kit (leica biosystems newcastle ltd ., newcastle, uk) 10 minutes quench endogenous peroxidase activity and washed 3 times in bond wash solution (leica biosystems newcastle ltd ., after that, sections were incubated for 15 minutes with 1: 100 dilution of monoclonal antibody to ck 4 (leica biosystems newcastle ltd .,, newcastle, uk), 1: 50 dilution of monoclonal antibody to ck 16 (leica biosystems newcastle ltd ., newcastle, uk) and 1: 200 dilution of monoclonal antibody to involucrin (leica biosystems newcastle ltd ., newcastle, uk). After washing the sections 3 times with bond wash solution for 1 minute, we incubated the sections for 8 minute with the post primary in bond polymer refine kit . After wash with bond wash solution and for an additional 8 minutes with polymer in bond polymer refine kit . After washing with bond wash solution, 3,3'-diaminobenzidine tetrahydrochloride (dab) in bond polymer refine kit for 10 minutes was used to visualize the peroxidase activity and hematoxylin was used to counterstain . All operate with bond - max auto immuno stainer (leica biosystems melbourne pty ltd ., vic, australia). All specimens were independently analyzed by two independent observers who were blinded to the specimen information . Both the basal, suprabasal (upper spinous, lower spinous and granular) and corneal layers of the epitheliums of normal ras and cholesteatoma were analyzed for positive staining . The positive - staining rates were determined in the areas where the full thickness of the epidermis could be clearly seen, and we designated cells that stained brown in the cytoplasm as positive . The results for the specimen training were graded as negative (-), weakly positive (+) if the percentage of positive cells was under 10%, moderately positive (+ +) if 10 - 70%, and strongly positive (+ + +) if over 70% . After the identification of well - stained areas under a light microscope at 100-fold magnification, we calculated the average percentage of three different spots at 400-fold magnification . Pearson's test was applied for the statistical analysis using spss ver . 17.0 (spss software, spss inc ., chicago, il, usa) program, and a p - value <0.05 was defined as a statistically significant difference . Immunohistochemical staining was performed to evaluate the expression of involucrin, filaggrin, ck 4, ck 10 and ck 16 . The tissues were fixed in 10% buffered neutral formalin solution and embedded in paraffin . For immunohistochemical staining, 4 m - paraffin embedded tissue sections were prepared on leica plus slide (leica biosystems richmond, inc ., the tissue sections were deparaffinized 3 times in dewax solution (leica biosystems newcastle ltd ., newcastle, uk) for 1 minute at 72, and then treated 4 times in 100% ethyl alcohol 1 minute respectively, prior to rehydration with distilled water . Antigen retrieval was performed by heating the slides for 15 minute in epitope retrieval solution 1, 2 (leica biosystems newcastle ltd ., newcastle, uk) and by cooling for 12 minutes . The slides were then treated with 3% h2o2 in bond polymer refine kit (leica biosystems newcastle ltd ., newcastle, uk) 10 minutes quench endogenous peroxidase activity and washed 3 times in bond wash solution (leica biosystems newcastle ltd ., after that, sections were incubated for 15 minutes with 1: 100 dilution of monoclonal antibody to ck 4 (leica biosystems newcastle ltd .,, newcastle, uk), 1: 50 dilution of monoclonal antibody to ck 16 (leica biosystems newcastle ltd ., newcastle, uk) and 1: 200 dilution of monoclonal antibody to involucrin (leica biosystems newcastle ltd ., newcastle, uk). After washing the sections 3 times with bond wash solution for 1 minute, we incubated the sections for 8 minute with the post primary in bond polymer refine kit . After wash with bond wash solution and for an additional 8 minutes with polymer in bond polymer refine kit . After washing with bond wash solution, 3,3'-diaminobenzidine tetrahydrochloride (dab) in bond polymer refine kit for 10 minutes was used to visualize the peroxidase activity and hematoxylin was used to counterstain . All operate with bond - max auto immuno stainer (leica biosystems melbourne pty ltd . All specimens were independently analyzed by two independent observers who were blinded to the specimen information . Both the basal, suprabasal (upper spinous, lower spinous and granular) and corneal layers of the epitheliums of normal ras and cholesteatoma were analyzed for positive staining . The positive - staining rates were determined in the areas where the full thickness of the epidermis could be clearly seen, and we designated cells that stained brown in the cytoplasm as positive . The results for the specimen training were graded as negative (-), weakly positive (+) if the percentage of positive cells was under 10%, moderately positive (+ +) if 10 - 70%, and strongly positive (+ + +) if over 70% . After the identification of well - stained areas under a light microscope at 100-fold magnification pearson's test was applied for the statistical analysis using spss ver . 17.0 (spss software, spss inc ., chicago, il, usa) program, and a p - value <0.05 was defined as a statistically significant difference . Involucrin was expressed in upper spinous, granular and corneal layers of ras and cholesteatoma . In upper spinous layer, involucrin expression was moderately positive in most of ras but strongly positive in all cholesteatomas (p=0.000). In granular layer, involucrin expression is strong in all ras and cholesteatoma . In corneal layer, cholesteatoma showed moderately or strongly positive expression of involucrin, compared with negative expression in most of ras (p=0.000)(table 1)(fig . Filaggrin was expressed in granular and corneal layers of ras and choelsteatoma . In granular layer, filaggrin expression was negative or weakly positive in most of ras, but weakly or moderately positive in cholesteatoma (p=0.001). In corneal layer, filagglin expression was strongly positive in most of cholesteatoma but not in ras (p=0.001)(table 1)(fig . Ck 4 was expressed in basal layer of ras with but not in cholesteatoma (p=0.000). In suprabasal layer both ras and cholesteatoma were not positive to ck 4 in corneal layer (table 2)(fig . Half of cholesteatoma showed moderately or strongly positive expression at suprabasal layer (p=0.006)(table 2)(fig . Ck 10 was homogenously strong - positive in all suprabasal and corneal layers of ras . While cholesteatoma showed negative expression in lower spinous layer (p=0.000), moderately to strongly positive in upper spinous layer (p=0.078), and strongly positive in granular layer . In corneal layer, ck 10 expression was weaker in most of the cholesteatoma than ras (p=0.015) (table 3)(fig . Involucrin was expressed in upper spinous, granular and corneal layers of ras and cholesteatoma . In upper spinous layer, involucrin expression was moderately positive in most of ras but strongly positive in all cholesteatomas (p=0.000). In granular layer, involucrin expression is strong in all ras and cholesteatoma . In corneal layer, cholesteatoma showed moderately or strongly positive expression of involucrin, compared with negative expression in most of ras (p=0.000)(table 1)(fig . Filaggrin was expressed in granular and corneal layers of ras and choelsteatoma . In granular layer, filaggrin expression was negative or weakly positive in most of ras, but weakly or moderately positive in cholesteatoma (p=0.001). In corneal layer, filagglin expression was strongly positive in most of cholesteatoma but not in ras (p=0.001)(table 1)(fig . Ck 4 was expressed in basal layer of ras with but not in cholesteatoma (p=0.000). In suprabasal layer both ras and cholesteatoma were not positive to ck 4 in corneal layer (table 2)(fig . Half of cholesteatoma showed moderately or strongly positive expression at suprabasal layer (p=0.006)(table 2)(fig . 2). Ck 10 was homogenously strong - positive in all suprabasal and corneal layers of ras . While cholesteatoma showed negative expression in lower spinous layer (p=0.000), moderately to strongly positive in upper spinous layer (p=0.078), and strongly positive in granular layer . In corneal layer, ck 10 expression was weaker in most of the cholesteatoma than ras (p=0.015) (table 3)(fig . Middle ear cholesteatoma is defined pathologically as the abnormal existence of keratinized squamous epithelium in the middle ear . Their common clinical hallmarks are invasion, migration, hyperproliferation, altered differentiation, aggressiveness and recidivism.11) and modern technologies using immuohistochiemistry, in situ hybridization, polymerase chain reaction, microarrays et al were applied to improve understating of the biologic properties of cholesteatoma.11) unlike normal skin, which has the karatinocytes proliferation capacity only in the basal cell layer of the epidermis, many studies for proliferative activity of cholesteatoma reported basal cell hyperplasia and appearance of suprabasal cells with new proliferation capacity . Normal keratinocytes that have lost the ability to divide move toward the suprabasal cell layers and terminally differentiated keratinocytes are anucleate and contain abundant cross - linked keratin filaments forming a cornified protein envelop . Epidermal homeostasis is maintained by a delicate balance between proliferation and terminal differentiation rate.1) in cholesteatoma, ki-67, a marker of cell proliferation, positive cells have been significantly observed in the suprabasal layer12) and proliferating cell nuclear antigen positive cells have been detected in the suprabasal and parabasal layer as well as in the basal layer of cholesteatoam epithelium.13) epidermal growth factor receptor also highly expressed in basal and suprabasal keratinocytes in cholesteatoma.14) cks used in this study are intermediated filaments proteins that are exclusively present in epithelial cells . The expression of different types of cks (numbered 1 - 20) varies with the type of epithelium and the stage of differentiation.9) according to the study of kakoi, et al.,15) the expression of cks 6, 16, and 19 in cholesteatoma epithelium showed hyperproliferative pattern . Patterns of the terminal differentiation of cks 1, 5, 10 and 14 in cholesteatoma were basically the same as those in skin . According to kim, et al.,16) ck expression correspond well with the state of keratinocyte proliferation, migration and differentiation . The result of their study using an animal model supports the concept that the normal keratinizing epithelium of the tympanic membrane undergoes certain changes as it forms a cholesteatoma in the external canal and middle ear . An increased expression of ck 5/6 and ck 13/16 is found in the suprabasal epithelial layer of the external auditory canal can be an evidence that cholesteatoma shows increased keratinocyte proliferation . Ck 4 is marker for non - keratinizing epithelia and indicates an altered differentiation and migration of keratinocytes.10) ck 10 is normally localized in the suprabasal layer of the epidermis and it's expression corresponded to the extent of differentiation within keratinocytes, so it can be a marker for differentiatioin.10,17) ck 16, a marker of proliferation, is partially expressed in basal layer of normal epidermis, but expressed in suprabasal layer in many epidermal proliferative diseases.16,18) our study showed ck 4 was expressed in basal layer of all ras . But in cholestetoma, there was no expression in basal layer while 33.3% expression in suprabasal layer . Ck 10 was strongly and homogenously expressed in all suprabasal and corneal layers of normal ras . On the other hand, in cholesteatoma ck expression was getting stronger from lower to upper layer . This pattern of' shifting to surface layer' presumed immature keratinocyte differentiation, and also presumed different velocity of differentiation, namely more faster in upper layer, compared with balanced differentiation of normal skin . Some other studies support our results,19,20) but kim, et al.16) reports ck 10 expression may be affected by the extent of cholesteatoma and the severity of disease . Ck 16 was occasionally expressed in only basal layer of normal skin, but strong expressed suprabasal layer in cholesteatoma . Hyperkeratosis was resulted from not only the hyperproliferation of basal and suprabasal layer, but also early differentiation of suprabasal layer in process of terminal differentiation from basal layer to corneal layer . Involucrin is the major protein component of the cross - linked envelope synthesized by maturing cells of human stratified squamous epithelia with a molecular weight of 140 kd . It is detectable in granular and upper spinous layer in normal skin, but not in both basal and corneal layer, so serves as a marker for the intermediate stage of squamous differentiation.5,6,21) stammberger, et al.21) reported that in cholesteatoma, involucrin was localized in the suprabasal cells, and particularly in spinous cell, and it appeared earlier than in ear canal skin . A larger amount of involucrin was present in cholesteatoma (60%) than in external ear canal skin (29%) and in normal skin (25%) in chao's study.22) park, et al.23) presented that involucrin was highly expressed in deep meatal skin as well as cholesteatoma than ras, which may support the migration theory of cholesteatoma pathogenesis . In our study, but in upper spinous layer involucrin was strongly expressed in all cholesteatomas than normal skin . In corneal layer, cholesteatoma showed highly positive expression of involucrin, compared with negative expression in most of ras . This results presumed that hyperdifferentiation and hyperkeratosis might be resulted from early differentiation of upper spinous layer . Filaggrin, a histidine - rich proteins, is a component of kertohyalin that is formed in differentiating cells . During formation of cornified cells, the basic filaggrins aggregate keratin filaments into large insoluble macrofibrils, conferring flexibility to the stratum corneum . Thus filaggrin can be a marker of epidermal differentiation.7,8) commonly, filaggrin was expressed in corneal layer of normal skin, whereas in cholesteatoma, it was expressed more in corneal and granular layer.7,21,23) our immunohistochemical study also got a similar results . Filaggrin overexpression in granular and corneal layer presumed that early differentiation may induce the typical hyperkeratosis of cholesteatoma . Based on these results, it can be suggested that early differentiation of suprabasal layer, especially upper spinous and granular layers in cholesteatoma, may lead to hyperdifferentiation and hyperkeratosis . Different expression of cks, compared with ras, possibly indicates the altered differentiation and hyperproliferation of cholesteatoma . Overexpression of involucrin and fillagrin presumed that early differentiation of suprabasal layer, especially upper spinous and granular layers in cholesteatoma, may lead to hyperdifferentiation and hyperkeratosis . Suprabasal expression of ck 4 and 16 and pattern of shifting to surface layer in ck 10, compared with ras, possibly indicate the altered differentiation and hyperproliferation of cholesteatoma.
We report a case of a 43-year - old male who sustained an electrical shock injury following which he fell from a height of around 4 feet . Laboratory investigations revealed the patient had extremely low levels of vitamin d (11.1ng / ml). We report this case as a rare etiological combination of hypovitaminosis d and electrical injury causing bilateral neck of femur fracture . Such injuries should be diagnosed at the earliest and goal of treatment should be to preserve both the hip joints . Early management would avoid potential complications like non - union and avascular necrosis . The reasons behind such injuries are varied . Electrical shock forms an exceedingly rare cause of bilateral neck of femur fracture . The reason for such an injury is due to violent unopposed muscle contractions rather than the trauma of fall per se . We report a case of simultaneous bilateral fractures of neck of femur occurring due to a rare etiological combination ofhypovitaminosis - d and electrical shock injury . A 43-year - old male patient presented to the emergency department with a fall from a height of 4 feet . He gave a history of charging his mobile, standing on a shelf of four feet height following which he suffered an electrocution injury . He fell down from the shelf, after which he was unable to stand up on his feet or move his legs due to pain . On examination, 1). Investigations revealed his vitamin d levels were low (11.1ng / ml). The patient was taken up for surgery, closed reduction and internal fixation by 6.5 mm cannulated cancellous screws . Surgical technique: pre - operative x - ray showing bilateral transcervical neck of femur fracture . The fracture was reduced by leadbetter s technique and reduction was maintained using traction table . Using the lateral approach, the tensor fascia lata was dissected and the area 2.5 cm distal to the vastus ridge was chosen for inserting three 6.5 mm cannulated cancellous screws with washers . After screws were inserted, reduction was checked again with the aid of c - arm . The left hip was fixed by the inverted triangle pattern of screw fixation, whereas the right hip was fixed with upright triangle pattern of fixation (fig . The patient was taught static quadriceps exercises and active knee and ankle range of movements 5 days after surgery . Patient was ambulated using a four stand walker at the end of 12 weeks . At the end of 16 weeks, patient was independently ambulatory and had good range of movements ofboth the hips (fig . 3, 4, 5) post - operative x - ray showing fixation of bilateral neck of femur with 6.5 cannulatedcancellous screws post - operative active bilateral hip flexion . Bilateral active bilateral hip abduction . Less than ten cases have been reported in our review of literature [1 - 6]. Most authors equivocally attribute this unique injury due to unopposed violent contractions of the musclesrather than the fall per se . In 1960, powell explained that the fractures occur specifically during the tonic phase of contractions of the muscles . This is evident by the fact that our patient fell from a height of less than 4 feet . This is not a sufficient force to cause fracture of bilateral neck of femur . In 1956, andreini suggested that simultaneous contraction of pertrochanteric muscles is the main cause of fractures around the hip following an electrical shock injury . He explained that only these muscles can apply their force irrespective of the position of pelvis and femur . He went on to conclude that if a person sustains an electrical injury when his legs are adducted, it would lead to central dislocation of bilateral hip joints . When the legs are abducted, it would lead to bilateral neck of femur fractures . In 1938, satta suggested that men are more prone to bilateral neck of femur fractures following an electrical shock due to the fact that muscles are stronger and well - toned as compared to women . Bone is the poorest conductor of electric current among all the living tissues in our body . Consequently, it provides the greatest resistance to flow of electric current resulting in it absorbing all the energy of the conducted current . In experimental studies, it has been observed that muscle contractions result from direct current of at least 20maand alternating current of 10ma . Authors have suggested that electroconvulsive therapy, drug induced epilepsy, chronic renal failure and metabolic conditions like hypocalcemia, osteomalacia are other causes of similar injuries in order of their frequency of presentation [8 - 15]. In 1947, santagali explained that bilateral neck of femur fractures tend to occur in metabolic conditions like osteomalacia and osteoporosis . Our patient had very low levels of serum vitamin d, which could also contribute to the grievous nature of the injury . Accidental electrical injuries are rare and often the patient is unconscious or in a confused state of mind . Initial sequelae of electric shock include thermal myonecrosis which leads to acute renal failure which complicates to cardiac arrest or cardiac arrhythmias . Hence, in the emergency room (er), the patient must be critically screened for cardiac and renal anomalies . Electrocardiogram with renal function tests with 24 hour monitoring is usually sufficient to rule out such complications [18, 19, 20]. Screening of pelvis, spine and shoulder is essential in order to avoid missing these rare injuries [8,16, 17]. In our review of literature, posterior shoulder dislocations, fractures of the proximal humerus and fracture of scapula have been reported following electric shock injuries due to vigorous muscle contractions around the shoulder[18, 21, 22]. In the forearm, galeazzi and distal radius fractures have been reported [18, 23, 24]. In the spine, l4 burst fractures and if the patient is conscious, a thorough clinical examination should suffice to rule out the above mentioned injuries . In our case, the patient was conscious and unable to lift both legs with painful range of movement of both hips . Hence, it was relatively easy to confirm the diagnosis after radiological studies . If the patient is not conscious, it becomes critical to screen the spine, pelvis and shoulders . Hip preservation should be the goal of treatment in young patients such as in our case . We took a decision of fixing both the femurs with three cannulated cancellous screws each . Dynamic hip screw can also be used as an alternative to treat such injuries . In older patients with a sedentary life style, bilateral hemiarthroplasty anesthetic complications like difficult intubation due to spasm or persisting cardiac abnormalities like arrhythmias are usually encountered . In such cases, surgery is deferred until the patient is stabilized . In our case complications like non - union and avascular necrosis are very common if neglected and hence the need to diagnose such injuries at the earliest [7, 8]. He was ambulatory and had good range of movements of the hip, knee and ankle to continue his normal life . We report this case as a rare etiological combination of hypovitaminosis d and electrical injury causing bilateral neck of femur fracture . Such injuries should be diagnosed at the earliest and goal of treatment should be to preserve both the hip joints . Early management would avoid potential complications like non - union and avascular necrosis the goal of the treatment should be to preserve both the hip joints with prior importance in treating hypovitaminosis - d, which would help in early healing of fracture and also prevent further pathlogical fractures following trivial trauma.
Psoriasis is a chronic skin disease that causes distress and morbidity, affecting approximately 2% of the population with an emotional, social, and physical impact.1,2 nail involvement has long been recognized as a common manifestation of psoriasis, occurring in up to 50% of patients, with an estimated lifetime incidence of 80%90%.3 its impact on quality of life is well known, since nail dystrophies are often highly visible, causing embarrassment for the patient and considerable disability in many cases.4,5 the most common clinical sign in nail psoriasis is pitting, which occurs in almost 70% of patients, followed by onycholysis.6 other nail manifestations associated with psoriasis are splinter hemorrhages, leukonychia, red spots in the lunula, nail plate crumbling, salmon patches, and hyperkeratosis.1 before starting an appropriate therapy for nail psoriasis, the severity of nail changes and the extent of skin and/or joint involvement should be considered . If a patient has extensive skin psoriasis accompanied with nail changes, systemic therapy will be appropriate, while for a patient who has predominant nail psoriasis with mild or no skin involvement, topical treatments would be an ideal initiating option for therapy.7 the choice of the topical formulation to be used depends on the presence of nail bed and/or nail matrix disease . Many products have been used in the topical treatment of nail psoriasis; corticosteroids and vitamin d analogs are still the most frequently used topical therapies found in literature.8 however, none of them have been fully satisfactory, thus determining a real clinical need for new therapeutic options . A product currently on the market, consisting of a hydro - soluble nail lacquer and containing hydroxypropyl - chitosan (hpch), horsetail extract (equisetum arvense), and methylsulfonylmethane, as its main ingredients, has shown its effectiveness in the management of nail splitting and nail brittleness.9 in a previous, preliminary study, we reported the effectiveness of this nail lacquer on signs of dystrophy in psoriatic nails,10 where the hpch nail lacquer was able to improve clinical signs such as pitting, leukonychia, and onycholysis (ie, the most common manifestations of nail psoriasis). The present placebo - controlled study was designed to confirm whether the strengthening and hardening properties of this product could improve the structure of the nail plates on psoriatic nails . The study was a randomized, single - center, double - blind, placebo controlled, parallel - group trial to evaluate the effects and tolerability of an hpch nail lacquer on nail psoriasis (the lacquer genadur, also known as ecocel, sililevo, betalfatrus, psoriatec, and kitolac; polichem sa, lugano, switzerland; it is also known as veralac distributed by valeo pharma inc ., it was approved by the independent ethics committee of the azienda policlinico umberto i, rome, italy, and was performed at the department of dermatology of the sapienza university, rome, italy . A total of 87 patients of both sexes and aged 1680 years were enrolled after providing written informed consent . Patients affected by mild to moderate nail psoriasis of the nail matrix and/or of the nail bed in at least one fingernail, with a clinical diagnosis of psoriasis at least 6 months previously, were randomly allocated in a 1:1 ratio to one of the study groups corresponding to the hpch nail lacquer or to placebo (which was the vehicle of the hpch nail lacquer). The main exclusion criteria were the use of any systemic treatment for the cutaneous lesions in the previous 3 months, any other topical product (drug or cosmetic) on any nail in the 4 weeks before the baseline visit, and a positive mycology test . The patients were treated once daily for 24 weeks on all fingernails in the evening at bedtime . The clinical features of nail psoriasis were evaluated every 4 weeks using the modified nail psoriasis severity index (mnapsi) on the target nail defined at baseline . According to the mnapsi definition,11 the target nail is divided into four quadrants, and to each quadrant, a score between 0 and 3 (0= none, 1= mild, 2= moderate, 3= severe) is given for each parameter of the nail matrix (presence or absence of pitting, leukonychia, red spots on the lunula, and plate crumbling) or the nail bed disease (salmon patch, onycholysis, hyperkeratosis, and splinter hemorrhages). The total score, resulting from the sum of all the partial scores, may vary between 0 (no signs) and 96, derived from 4 (quadrants) 8 (parameters) 3 (severe disease). The primary end point was the clinical cure rate, which was defined as an mnapsi score 4 at the end of treatment . A further clinical end point was the proportion of patients who achieved a 50% reduction in the score (mnapsi-50) compared to baseline value (ie, an improvement 50% of the baseline mnapsi score). The acceptability of the product was assessed by the patient at the end of the study according to a five - point scale: 1= very good; 2= good; 3= fair; 4= poor; 5= very poor . Data were processed by means of the program sas for windows, release 9.2 (sas institute inc ., descriptive statistics were performed, including the mean, standard deviation, standard error, minimum value, median, and maximum value for continuous variables . According to the last observation carried forward approach,12 the last observed value of the mnapsi score was used as an end point value in the event that patients prematurely withdrew from the study (for any reason). The significance level was declared as a p - value <0.05 (two - tailed test). The superiority of the test product versus placebo was tested for efficacy parameters in the intention - to - treat (itt) population by means of fisher s exact test . The same model was applied on the per protocol (pp) population as a supportive efficacy analysis for the aforementioned parameters . The difference in patients judgments on treatment acceptability was analyzed by means of the chi - square test . A total of 87 patients were randomized: 43 in the hpch nail lacquer group and 44 in the placebo group . Eighty - one out of 87 patients were considered evaluable for the itt efficacy analysis since six patients (three in each group) had no postbaseline assessments . The two groups were similar with respect to their demographic characteristics (table 1). The clinical cure rate, which was defined as the primary end point and evaluated every 4 weeks, showed that after 24 weeks (end of treatment) there was a statistically significant superiority of the hpch nail lacquer compared to placebo in both the itt (fisher s exact test, p=0.0445) and pp populations (fisher s exact test, p=0.0437). To be precise, in the itt dataset, 55% (22/40) of patients treated with the hpch nail lacquer versus 31.7% (13/41) of the placebo patients were cured after 24 weeks (figure 1). The superiority of the test product over placebo was already evident after 16 weeks in 35.0% (14/40) of the hpch nail lacquer treated patients versus 14.6% (6/41) of the placebo patients (fisher s exact test, p=0.0415; itt population). The difference was higher when the pp dataset, including all patients who completed the trial according to the protocol, was considered: 67.7% (21/31) of patients treated with the hpch nail lacquer and 40.6% (13/32) of patients randomized to the placebo had become cured at the end of the treatment period (figure 2). The analysis of mnapsi-50 indicates a trend of superiority of hpch nail lacquer compared to placebo (figure 3) throughout the 24-week treatment course . Moreover, the clinical improvement observed in the test product group had already been confirmed after 12 weeks of treatment, in fact the mnapsi-50 score was statistically superior in comparison to the results obtained after 8 weeks of treatment (fisher s exact test, p<0.05). The improvement of clinical signs of nail psoriasis at the end of treatment is documented in figures 4 and 5 . The level of acceptability of the products by the patients was also evaluated in the itt population . A statistically significant difference was observed between treatments, in favor of the hpch nail lacquer (figure 6) at the end of the treatment period (chi - square test, p=0.0265). In detail, the level of acceptability was assessed as good or very good by 96.8% of patients in the hpch nail lacquer group (30/31) compared to 78.1% (25/32) of placebo - treated subjects . A case report of a patient who had not been cured at the end of the treatment period is described as follows . The patient, encouraged by the result obtained after 6 months of treatment, decided to continue the product application, and after 27 months, the nail psoriasis was cured without using any systemic or other topical nail treatments (figure 7). Eight patients reported nonserious adverse events, six in the placebo group and two in the hpch nail lacquer group . No adverse events were assessed by the investigator as being related to the study treatment . Psoriasis of the nail is associated with disability, as the nails are weak and often painful in this condition; this has remained an unsatisfied medical need over the last 2 decades.4,13 the choice of treatment for nail psoriasis depends on the clinical features of the disease, as well as on individual factors for each patient . If the associated skin psoriasis is not systemically treated, or if nail psoriasis is the sole manifestation of the disease, topical therapies are the appropriate first choice . The current treatment options for nail psoriasis are poorly efficacious, and patients need long - term treatments in order to obtain any clinical benefit.5,14 the situation has not greatly changed after the introduction of biologics in the management of patients with psoriasis.13 biologic agents have demonstrated the best outcome compared with other available therapeutic methods; however, the relatively higher social costs and greater risks of adverse effects remain a serious concern . Hence, systemic treatment should be reserved for a minority of patients with severe skin psoriasis and/or joint involvement.7,13 at the moment, topical treatment options are limited or absent due to the great difficulty experienced in delivering the drug to the site of action, and also due to the fact that the commercially available products are usually in the form of creams or gels, which are unsuitable to remain in contact with the nails for a sufficient period of time, unless they are applied by means of complicated occlusive or semiocclusive medications.1517 topical therapy for nail psoriasis would be very welcome if nail lacquers become available that demonstrate activity in this condition . We previously reported the ability of this hpch nail lacquer to decrease signs of dystrophy in psoriatic nails,10 although that study was neither randomized nor blind, and it had only a preliminary value . The present randomized, placebo - controlled, double - blind trial could be considered as one of the most powerful clinical studies performed to date, and the results confirm the effectiveness of hpch nail lacquer applied for 24 weeks in nail psoriasis, with the test product proving to be statistically superior to placebo . Currently, no other powered study on the management of nail psoriasis that compares a topical medical device to placebo has been published.18 the nail psoriasis severity index (napsi) has been developed as an objective and reproducible tool to standardize the treatment outcome in nail psoriasis treatment . Specifically, the current napsi system, which uses a 32-point scale for the target nail without taking into consideration disease severity, may lack the sensitivity to reflect meaningful clinical improvement.19 in 2005, the mnapsi, with scores ranging between 096 for the target nail, and using a qualitative gradation of severity for each parameter, (from 03) in each quadrant, was proposed by parrish et al.11 this alternative evaluation index was developed in order to increase the sensitivity of the napsi to reflect the response to therapy of the disease . Nevertheless, the simple decrease of the aforementioned scores may not reflect the real clinical outcome and the complete or almost complete resolution of the signs and symptoms of nail psoriasis . For this purpose, in our investigation, we proposed an mnapsi cut - off score of 4 based on the clinical observation that a value 4 does not cause disability and shows a robust clearing of the psoriatic nail lesion . This value is consistent with the published literature.19 in fact, rigopoulos et al20 have already defined that a value between 34 indicates an almost complete resolution of psoriatic nail involvement . This evaluation is the current benchmark used in most clinical trials of psoriasis.21 it could be hypothesized that a 50% reduction in the mnapsi score represents a meaningful change in the clinical outcome . Although the clinical benefit of the treatment was already evident after 12 weeks, the 24-week treatment course is the minimum period required to demonstrate evidence of the treatment s meaningful effects . No differences in terms of the safety profile between the product and the placebo were found during the study . In addition, the clinical setting suggests that the longer the treatment period is, the greater the efficacy of the hpch nail lacquer . However, considering that a treatment lasting longer than 6 months for the resolution of nail psoriasis is justified by the high safety and tolerability profile of the product, further experiments should be designed in order to confirm the reported results . In conclusion, considering the poor clinical outcome of the available systemic and topical treatments for nail psoriasis, hpch nail lacquer is a valid, effective, and safe option that can be used to decrease the signs of nail dystrophy in psoriatic patients.
These studies were approved by institutional review boards at the national institutes of health (nih) and washington university . A 28-year - old woman with job syndrome pathologic examination of a bronchoalveolar lavage (bal) sample showed scattered cells, primarily columnar bronchial cells, with cytomorphologic changes reminiscent of bk polyomavirus (bkpyv)infected decoy cells . The cells stained positive with pab416, a monoclonal antibody against the sv40 large t antigen . The patient had bkpyv viremia (8.1 10 copies / ml) and viruria (6.9 10 copies / ml). The bal sample was weakly positive for bkpyv by pcr (<250 copies / ml) and negative for jc polyomavirus . Nonenveloped virions were purified from the bal sample by using ultracentrifugation with optiprep (#d1556; sigma - aldrich, st . Dna was extracted from the virion preparation and subjected to random - primed rolling circle amplification (rca) and restriction enzyme digestion, which yielded 2 strong bands . The bands were cloned and identified as wupyv by using sanger sequencing . The complete genomic sequence of the isolate, designated j1 (genbank accession no . Kj643309), was confirmed by using miseq analysis (illumnina, san diego, ca, usa) of the rca product . A second wupyv variant with 2-nt polymorphisms and a single base insertion was also detected in the rca product . We developed an immunohistochemical (ihc) assay to detect the wupyv viral protein 1 (wu - vp1) by using an igg2b designated nn - ab06 . Recombinant histidine - tagged wu - vp1 protein was generated by expressing wu - vp1 (genbank accession no . Abq09289) in escherichia coli from a gateway pdest17 plasmid (life technologies, carlsbad, ca, usa) and purifying the protein by using an affinity ni - nta column (pierce biotechnology, rockford, il, usa). After generation of hybridomas, we identified clones producing antibody against wu - vp1 by elisa and immunoblot . Clones that cross - reacted with ki polyomavirus vp1 (ki - vp1) were identified by elisa with glutathione s - transferase tagged ki - vp1 (2) and eliminated . To generate positive control cells for ihc assay optimization, we transfected 293 t cells with plasmid pdest26-wu - vp1 (life technologies). A subset of cells was fixed in 10% neutral - buffered formalin and embedded in paraffin . Ihc testing was performed by deparaffinizing slides in xylene and rehydrating them in a series of ethanol solutions . After treating slides with 3% hydrogen peroxide, antigen was retrieved in citrate buffer, ph 6.0 (10 mmol / l citric acid, 0.05% tween 20) in a pressure cooker (pc625; nesco, two rivers, wi, usa) for 3 min on the high setting . Slides were blocked in 1.5% normal horse serum (#s-200; vector laboratories, burlingame, ca, usa) and incubated with nn - ab06, then with biotinylated anti - mouse igg (ba-2000; vector laboratories). Biotin complex kit (#pk-6100; vector laboratories) and (3,3-diaminobenzidine) (#sk-4100; vector laboratories), we counterstained tissues with hematoxylin . A serial section of the same cell block stained with an isotype - matched antibody (#557351, mouse igg2b: bd biosciences, san jose, ca, usa) (figure 1, panel b) and mock transfected cells stained with nn - ab06 (figure 1, panel c) showed negative results . Western blotting was performed as an independent means of evaluating specificity of nn - ab06 (11). Nn - ab06 reacted with wu - vp1 protein lysate but not with ki - vp1 lysate, which is the most closely related virus to wupyv . Wu polyomavirus antigen in bronchoalveolar lavage specimens from lungs transplanted into a recipient (28-year - old woman) with job syndrome . Immunohistochemical analysis of 293 t cells transfected with pdest26-wu virus protein 1 and stained as follows . D) bronchoalveolar lavage specimen stained with nn - ab06 showing prominent dark staining of cells with enlarged nuclei and a ground glass appearance characteristic of viral cytopathic changes (arrows). Original magnifications 400 in panels a c and 600 in panels d and e. we applied the wu - vp1 ihc assay to formalin - fixed, paraffin - embedded sections of the bal sample . Prominent dark staining of cells with enlarged nuclei and a ground glass appearance characteristic of viral cytopathic changes were observed (figure 1, panel d). Staining was not seen in serial sections stained with the isotype antibody (figure 1, panel e) or with no antibodies . Many wupyv - positive cells were cuboidal to columnar and showed other morphologic features consistent with respiratory epithelial cells . To determine their etiology, we developed a double immunofluorescence (dif) assay with a polyclonal antibody against wu - vp1 (2), designated nn - ab01, and a monoclonal antibody against cytokeratins (#m3515; dako, carpinteria, ca, usa). Deparaffinization and antigen retrieval were accomplished as noted above, and sections were blocked in superblock t20 (#37516; thermo scientific, waltham, ma, usa). To validate the assay, we performed immunofluorescence analysis with nn - ab01 on positive control 293 t cells expressing wu - vp1 . Several wu - vp1-positive cells were observed (figure 2, panel a); a serial section stained with preimmune serum at the same dilution showed a negative result (figure 2, panel b). Wu polyomavirus antigen in respiratory epithelial cells from lungs transplanted into a recipient (28-year - old woman) with job syndrome . Immunofluorescence of 293 t cells transfected with pdest26-wu virus protein 1 and stained with a) wu virus protein 1 polyclonal antibody (nn - ab01) or b) preimmune serum . C) double immunofluorescence with nn - ab01 (red) and a monoclonal antibody against cytokeratin (green) showing a double - positive cell from the bronchoalveolar lavage specimen . D) bronchoalveolar lavage specimen with multiple wu virus protein 1/cytokeratin double - positive cells . Original magnifications 100 in panels a and b, 600 in panel c, and 400 in panel d. for dif, bal sections were incubated first with the primary antibodies and then with fluorescently labeled secondary antibodies (#a10042 anti - rabbit-568 and #a10042 anti - mouse-488; life technologies). We observed cells positive for wu - vp1 and cytokeratin (figure 2, panels c, d), which identified these cells as epithelial cells . Of the 136 wu - vp1-positive cells, 77 (57%) were also cytokeratin positive . A serial section of the bal sample stained with an isotype - matched antibody to the cytokeratin antibody (#555746 mouse igg1; bd biosciences) and preimmune rabbit serum was negative . We hypothesized that the remaining 43% of wu - vp1-positive, cytokeratin - negative cells might be macrophages . However, a dif assay using nn - ab01 and an antibody against cd68 (#m0814; dako), a macrophage marker, showed wu - vp1 and cd68 single - positive cells but no double - positive cells . In addition, a stain with nn - ab01 and an antibody against cd45 (#m3515292; dako), a marker for hematopoietic cells, also showed negative results . Before this study, to our knowledge, no specific cell type had been identified as susceptible to wupyv infection . The presence of nuclease - resistant viral dna from the optiprep gradient and detection of wu - vp1, which is believed to be expressed concomitantly with dna replication (12), suggests that the cells were infected by wupyv and that the wupyv life cycle reached at least the stage of late gene expression . The clinical role of infection by wupyv is uncertain, given that the patient was not experiencing any recognizable symptoms . Although we attempted to identify a second population of wu - vp1-positive, cytokeratin - negative cells, the etiologic features of these cells remains uncertain . Our patient had job syndrome, a primary immunodeficiency not previously associated with polyomavirus susceptibility . It is possible that immunosuppressant medications, which include prednisone and tacrolimus, altered susceptibility to virus infection . Other human polyomaviruses are believed to exclusively cause disease in immunocompromised hosts this case suggests that immunosuppression might also play a role in wupyv infection and expands our understanding of wupyv biology.
Tennis elbow (te)also called lateral epicondylitis, epicondylosis, epicondylalgia or tendinopathy is a common disorder of the elbow with a prevalence of 13% in the general population and 7% in manual workers [8, 28]. Previous studies have suggested a prevalence of 3550% among tennis players [8, 28]. However, a recent prospective study in junior tennis players reported elbow injuries in 9% during the two studied years and found injuries to the ankle, shoulder or low back to be more common . Te is occurring most often in the age group of 4060 years except in tennis players who are generally younger and it affects men and women to the same degree [8, 20, 28]. In addition to age, risk factors for developing tennis elbow include repetitive and forceful motions of wrist and arm, participating in racket sports, using a faulty tennis playing technique and smoking tobacco . It has been claimed that conservative care leads to recovery in up to 90% of te patients within 12 years and that surgery is indicated in less than 10% of the cases [5, 6, 25]. However, recent studies of patients with elbow complaints, including te, in general practice report a less favourable prognosis [2, 17]. Bot and co - workers found that although 90% of all patients reported at least some improvement after 1 year of follow - up, only 13% of the patients reported full recovery at the 3-month follow - up and 34% at 12 months . In patients with persisting pain and disability, many different techniques have been described . However, at present no technique has been shown to lead to better results than the others . Few randomised studies have been reported, and many case studies are hampered by methodological shortcomings such as small study population, low percentage of follow - up and inclusion of cases with concomitant lesions in elbow, hand or shoulder . The purpose of this study was to evaluate short - term (median 18 months after surgery) and medium - term (median 4 years after the surgery) results after open lateral release in recalcitrant tennis elbow and to determine any prognostic factors . Eighty - nine patients with 92 operated elbows met the criteria for inclusion in the study . Thus, 77 patients (87%), 38 male and 39 female, with 80 operated elbows were available for analyses . The median patient age at the time of surgery was 46 years (range, 3464 years). Patients with a characteristic history of activity - related pain at the lateral epicondyle interfering with the activities of daily living refractory to conservative care for at least 6 months and a confirmatory physical examination that included palpable tenderness over the extensor tendon insertion, provoked pain with resisted wrist and third digit extension, normal range of movement of the elbow, normal neurological status, normal ligamentous laxity tests and no radiographic joint derangement were included in the study . Exclusion criteria were restricted range of movement, neurologic deficits, ligamentous instability, previous surgery in the elbow, fracture sequelae, chondral or osteochondral lesions, osteoarthritis or loose bodies . Clinical outcome was evaluated by the 11-item disability / symptom subset of disabilities of the arm, shoulder and hand outcome measure (dash) named quickdash . Data were collected before the operation and at the medians of 18 months (range, 636 months; short term) and 4 years (range, 36 years; medium term) postoperatively . We rated the quickdash outcome at the last follow - up as excellent (<20 points), good (2039 points), fair (4060 points) or poor (> 60 points). The patient s profession was classified as strenuous or non - strenuous with respect to the upper extremities . The surgery was carried out in an outpatient surgery unit in combined general anaesthesia and local anaesthesia subcutaneously with the patient placed supine with a standard technique similar to that described by verhaar and co - workers . The extensor origin was exposed, divided transversely close to its attachment on the lateral epicondyle and allowed to retract distally . The joint capsule was released along with the extensor origin, and a small incision was made through the synovial membrane allowing inspection of the joint . Decortication of the bone at the attachment site at the lateral epicondyle was performed with an osteotome . Rehabilitation consisted of early active range of motion and eventual return to full activity as tolerated . Heavy or repetitive manual work was discouraged for 6 weeks . The statistical analyses were made with the statistical package for the social sciences (spss inc ., chicago, illinois, usa) on a personal computer . As measures of central location and spread of data, repeated measures one - way anova was used to compare the quickdash scores at different points in time . Multiple regression was used to explore the relationship (regression model) between quickdash at the final follow - up (dependent variable) and a combination of possible predictor variables; baseline quickdash, sex, age, occurrence in dominant or non - dominant arm, duration of symptoms, sudden or gradual onset of symptoms, work - related cause, strenuous or non - strenuous work and/or occupation . Clinical outcome was evaluated by the 11-item disability / symptom subset of disabilities of the arm, shoulder and hand outcome measure (dash) named quickdash . Data were collected before the operation and at the medians of 18 months (range, 636 months; short term) and 4 years (range, 36 years; medium term) postoperatively . We rated the quickdash outcome at the last follow - up as excellent (<20 points), good (2039 points), fair (4060 points) or poor (> 60 points). The patient s profession was classified as strenuous or non - strenuous with respect to the upper extremities . The surgery was carried out in an outpatient surgery unit in combined general anaesthesia and local anaesthesia subcutaneously with the patient placed supine with a standard technique similar to that described by verhaar and co - workers . The extensor origin was exposed, divided transversely close to its attachment on the lateral epicondyle and allowed to retract distally . The joint capsule was released along with the extensor origin, and a small incision was made through the synovial membrane allowing inspection of the joint . Decortication of the bone at the attachment site at the lateral epicondyle was performed with an osteotome . The arm was rested in a sling for 2 weeks . Rehabilitation consisted of early active range of motion and eventual return to full activity as tolerated the statistical analyses were made with the statistical package for the social sciences (spss inc ., chicago, illinois, usa) on a personal computer . As measures of central location and spread of data, repeated measures one - way anova was used to compare the quickdash scores at different points in time . Multiple regression was used to explore the relationship (regression model) between quickdash at the final follow - up (dependent variable) and a combination of possible predictor variables; baseline quickdash, sex, age, occurrence in dominant or non - dominant arm, duration of symptoms, sudden or gradual onset of symptoms, work - related cause, strenuous or non - strenuous work and/or occupation . The median duration of symptoms was 13 months (range, 672 months). Twenty - six patients considered their work as the main cause of the elbow problem, whereas 2 patients related their problem to sporting activities . Twenty - three patients had occupations classified as strenuous according to haahr et al . . We did not observe macroscopic ruptures or other convincingly grossly pathologic changes in the extensor origin or internal derangement of the joint such as chondral or osteochondral lesions, osteoarthritis or loose bodies . Major complications such as deep infection, permanent nerve injuries or stiffness of the elbow were not observed . Superficial wound problem / infection was seen in three patients, and a postoperative haematoma was evacuated in one patient . In three patients, the mean quickdash was significantly improved compared with baseline both at the median 18-month and the median 4-year follow - ups (table 1). No significant difference was found in mean quickdash between the short - term and the medium - term follow - ups . An improvement of the quickdash at the final follow - up compared with the baseline was observed in 78 of 80 (97.5%) elbows . As excellent in 58 of 80 elbows; good in 7 elbows, fair in 11 elbows and poor in 4 elbows.table 1the mean value sd of the quickdash [from 0 (best) to 100 (worst)] before the operation and at the short- and medium - term follow - upsquickdashp valuepreoperative61 16median 18-month follow - up17 20p <0.001median 4-year follow - up18 19p <0.001 in comparison with preoperative data the mean value sd of the quickdash [from 0 (best) to 100 (worst)] before the operation and at the short- and medium - term follow - ups * in comparison with preoperative data we found a moderate correlation between the short - term and the medium - term results for the quickdash (r = 0.691; p <0.001). We found a weak correlation between the quickdash at the final follow - up (a high value denotes residual symptoms) and baseline quickdash (r = 0.388; p <0.001), acute occurrence of symptoms (r = 0.362; p <0.001), duration of symptoms (r = 0.276; p = 0.007), female gender of patient (r = 0.269; p = 0.009) and age of patient (r = 0.203; p = 0.04). We found no significant correlation between the quickdash at the final follow - up and affection of dominant (vs. non - dominant) arm, a work - related cause (as evaluated by the patient) or strenuous (vs. non - strenuous) work and/or occupation . The linear regression line equation was as follows: (quickdash at final follow - up) = 15.335 + 0.247 (baseline quickdash) + 17.845 (acute occurrence) + 0.388 (duration) + 4.057 (female gender) 0.440 (age) (p <0.001). Tennis elbow is generally believed to be caused by repetitive mechanical load of the elbow while using a forceful hand grip leading to an overuse injury of the extensor tendons insertion . The condition has often been called epicondylitis but histologic examinations have failed to demonstrate inflammatory cells . The pathogenesis is believed to be cumulative microtrauma exceeding the tissue s capacity for repair leading to a degenerative process characterised by disruption of tendon fibres, invasion of fibroblasts, disorganised collagen and vascular hyperplasia [5, 25]. Macroscopically, the findings reported by different authors vary greatly and include little or no grossly pathologic findings [3, 30, 33]; greyish, immature scar tissue which appears shiny, oedematous and friable; and partial or total rupture of the extensor tendon origin possibly reflecting different stages of a degenerative process . Swedish studies during the last decade have suggested that the pain in tennis elbow as in achilles and patellar tendinosis is caused by a so - called neurogenic inflammation mediated through neuropeptides such as substance - p and calcitonin gene regulated peptide [13, 23, 36]. Most surgical techniques aim to provide one or more of the following: relieve the stress at the tendons insertion by a release of the common extensor origin [19, 33]; removal of the degenerative tissue; or stimulating repair by decortication of bone at the insertion site [25, 33]. A lateral release is performed by an open, mini - open or percutaneous approach . The results after open surgery with recalcitrant te have been reviewed by meta - analyses recently [20, 24]. While the different studies constituting the meta - analyses cannot be directly compared, the surgical success rates for the open technique have been reported to be between 19 and 100% with a mean of 80.4% . Thus, the results of our study81% was rated as excellent or good at the final median 4-year follow - up seem to be in accordance with the typical outcome after open surgery . In the present study, the quickdash is a more efficient version of the dash outcome measure that appears to retain its measurement properties and can be used instead of the dash with similar precision in upper extremity disorders . The dash or quickdash scores have been used for evaluating the outcome after surgical treatment of te in several studies [10, 22, 32, 34]. Most commonly are removal of tissue with macroscopic degenerative changes and decortication of bone with an osteotome or by drilling . In the present study, decortication by an osteotome of a small area of bone at the insertion site of the extensor origin the rationale for decortication is the release of pluripotent stem cells that may accelerate the repair process . However, some studies suggest that decortication is not necessary for achieving repair and that it may even hamper the outcome by resulting in more postoperative wound bleeding, stiffness and pain [21, 37]. We observed only one case of postoperative hematoma requiring evacuation, but the design of our study does not permit any conclusion about the effect of decortication on the rate of complications or the outcome . Similar to the observation by others [9, 33], we found little or no macroscopic degenerative changes in the extensor origin . However, as a transverse division of the extensor origin was performed only superficial changes will be visible, and deeper tendinous ruptures may be overlooked [25, 33]. We do not consider this to be a problem as the surgical technique used in our study does not include removal of tendinous tissue . In outcome studies with an observation period averaging 2 years or more, a good or excellent outcome has been reported in over 80% of the patients by both the nirschl surgical technique or modifications of the latter that includes removal of degenerative tendinous tissue [11, 12, 25] and the hohmann extensor release without removal of degenerative tendinous tissue [27, 33]. Further, there is no correlation between the intensity of the histologic reaction and the clinical outcome . At present, it has not been shown that removal of degenerative tendinous tissue results in an improved clinical outcome [20, 24]. The age (median, 46 years; range, 3464 years), distribution of gender (38 male and 39 female) and percentage affection of dominant arm (71%) of our patients are similar to that of other studies on surgical treatment of te [25, 33]. The baseline symptoms and disability of our patients as evaluated by the mean quickdash (61)are also similar to that reported by others using the same set of questions [10, 22]. We found that high level of pain and disability at baseline (r = 0.388; p <0.001), acute occurrence of symptoms (r = 0.362; p <0.001), long duration of symptoms (r = 0.276; p = 0.007), female gender (r = 0.269; p = 0.009) and young age (r = 0.203; p = 0.04) are weak predictors of poor outcome, i.e. A high quickdash . Unfortunately, few previous studies analyse possible predictors of poor (or good) outcome after te surgery and it is difficult to find studies that support or refute our findings . High level of pain and disability at baseline [2, 15] and long duration of symptoms [2, 29] have previously been shown to predict worse outcome after conservative treatment of te . Worse clinical outcome in women has previously been observed after both conservative care, including physiotherapy [2, 35] and surgery . Many clinical studies on surgical treatment of te are hampered by shortcomings including retrospective design, low number of patients, loss of patients to follow - up, short - term follow - up period and inclusion of cases with concomitant procedures . In evaluating the coleman methodology score (cms) of studies on operative management of tennis elbow, karkhanis and co - workers found that only 9 of 45 studies reported on more than 60 elbows (which is the lower study size limit for the top score of the cms) and only five studies were sized 80 elbows or more . In the present study, 80 elbows (in 77 patients) we managed to follow - up 82% of the patients eligible for inclusion in the study . Karkhanis found that the mean follow - up time fluctuated from 12 to 96 months in 45 outcome studies on surgical treatment of tennis elbow . Whereas a long observation period is generally considered to strengthen the scientific value of a clinical study, most patients are just as interested in information about the short - term prognosis . Thus, it makes sense to include both a short - term follow - up and a medium- or long - term follow - up and to examine if a surgical result changes over time . In the present study, we evaluated the short - term (median 18 months) and medium - term (median 4 years) results of the treatment of recalcitrant te with release of the common extensor origin . Contrary to the results of verhaar et al . Who found a clinical improvement from 1 to 5 years postoperatively, no significant difference in outcome between the short - term follow - up and the medium - term follow - up was demonstrated in our study however, our first follow - up was done at a later point in time (medium 18 months) than in the study of verhaar et al . . The strengths of our study include the high number of patients, more than 80% follow - up, a single uniform surgical technique, no concomitant lesions, both short- and mid - term follow - ups, the use of a patient administered outcome score and registration of baseline symptoms / disability . The important limitations of our study are the lack of a control group and functional testing, e.g. Grip strength . We conclude that open lateral extensor release performed as outpatient surgery results in improved clinical outcome at both short- and medium - term follow - ups with few complications . At the final follow - up (at median 4 years postoperatively), 81% was rated as excellent or good a result that seems to be in accordance with the typical outcome after open surgery . High baseline disability, sudden occurrence of symptoms, long duration of symptoms, female gender and young age were found to be weak predictors of poor outcome.
Recent clinical research has provided evidence demonstrating that low - dose intravenous infusions of ketamine, a drug originally developed as an anaesthetic, can improve depressive symptoms within hours in subjects with treatment - resistant depression . Interestingly, the effects have been shown to last from a couple of days up to several weeks . This is a crucial step forward in the treatment of depression, among the greatest challenges that modern medicine has ever been forced to face, thought to affect up to 350 million people worldwide . The antidepressant medications available today exhibit low rates of treatment response, with only one in three people responding to their first prescribed medication and two in three people responding after trying numerous alternatives . More importantly, therapeutic effects display a response lag time of several weeks, a significant problem in those individuals who are particularly vulnerable to self - harm and suicide . For these reasons, there is a pressing need to identify novel antidepressant drugs that are fast acting and show better rates of response . Despite the promising rapid antidepressant action, ketamine has psychotomimetic and addictive properties that limit its potential widespread use as a fast - acting antidepressant drug . Indeed, ketamine has been shown to induce psychosis in healthy subjects and to exacerbate psychotic symptoms in individuals affected by schizophrenia, and has also been abused as a club drug' . Elucidating the molecular pathways via which ketamine mediates its antidepressant effects would facilitate the development of other pharmacological agents with similar beneficiary properties but without the unwanted side effects . We focus on three specific molecular mechanisms that are potentially involved in the antidepressant action of ketamine: increased neuroplasticity and synaptogenesis via enhancement of glutamatergic signaling, changes in immune function and (more preliminary) regulation of glycogen synthase kinase-3 (gsk-3) activity . Ketamine is classified pharmacologically as an n - methyl - d - aspartate (nmda) receptor antagonist . The first indication that the nmda receptor may be a useful target for antidepressant treatment came from observations that the anti - tuberculosis drug, cycloserine, a partial agonist of the glycine site of the nmda receptor, improved mood in those tuberculosis - affected patients who were also depressed . It subsequently took more than 30 years to develop the hypothesis that compounds altering nmda function could have antidepressant properties and therefore that glutamate, its main ligand, may be involved in the pathophysiology of depression . However, it has been suggested that other glutamatergic receptors may be involved in the action of ketamine . Indeed, when glutamate (l - glutamic acid), the major excitatory neurotransmitter in the nervous system, is released from presynaptic neurons, it can interact with different postsynaptic receptors: kainite, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (ampa), and nmda . Several findings support the involvement of both nmda and ampa receptors in the pathophysiology of major depression disorder (mdd) and in the mechanisms of action of antidepressants . Ketamine is able to increase extracellular glutamate levels in the prefrontal cortex (pfc) by inhibiting nmda receptor currents on gabaergic interneurones, in turn disinhibiting glutamate transmission . In particular, an increase in ampa / nmda receptor density ratio has been observed in the hippocampus of rats after ketamine treatment . Indeed, an enhanced glutamatergic activity transduced through ampa receptors rather than nmda receptors may be responsible for mediating the increased synaptic potentiation and activation of early neuroplastic genes observed upon exposure to the drug (described below in further detail). Of note, treatment with nbqx, an ampa receptor antagonist, has been shown to inhibit the antidepressant effects of ketamine in animal models of depression . Both ampa and nmda receptors are important in long - term potentiation and long - term depression, the two main neurobiological mechanisms which are responsible for mediating activity - dependent synaptic plasticity and re - modeling . Long - term potentiation has primarily been shown to induce dendritic spine growth, and enlargement of pre - existing spines and of the associated post - synaptic density proteins, all of which are observed upon ketamine exposure . Additionally, ketamine inhibits spontaneous nmda mini - excitatory post - synaptic currents caused by spontaneous glutamate release at rest . Interestingly, brain post mortem studies have described increased glutamate levels in individuals with mood disorders, while decreased hippocampal nmda receptors have been described in bipolar patients . Ketamine has also been shown to reverse the dendritic atrophy caused by chronic unpredictable stress exposure, a paradigm widely used to induce depression - like behavior in rodents . In such a model, a single dose of ketamine increased the number of spines on the apical dendrites of pfc layer v pyramidal neurons and also their function, as demonstrated by increases in serotonin- and hypocretin - induced excitatory post - synaptic currents . These changes occurred 2 h after administration of the drug and were sustained for up to 7 days, a time course comparable to that reported in clinical trials . Concomitantly, the reduced interest in both sucrose and food that followed chronic unpredictable stress was completely abolished . Bdnf has a central role in the neurotrophic theory of depression, which proposes that stress - related reduction in neurotrophic support, leading to the degeneration of limbic structures and, in particular, of the pfc and hippocampus, represents an important factor underlying the pathogenesis of depression . More specifically, reductions in bdnf, and of its high affinity receptor trkb, have been found both in the blood and the brains of patients affected by mood disorders; conversely, antidepressant drugs have shown to act, at least in part, through a potentiation of bdnf expression and its signaling . Bdnf has also been associated with the process of neurogenesis, thought to be impaired in stress and increased by antidepressants . Additionally, bdnf has been linked to synaptic re - modeling, being able to both induce and be induced by long - term potentiation . Animal models have shown that ketamine administration reduced immobility in the forced swim test, and this behavioral effect was coupled with increased bdnf protein levels in the hippocampus . Furthermore, circulating bdnf levels were increased after ketamine administration in treatment - resistant mdd patients concomitant with mood improvement . For example, treatment of primary neuronal cultures and hippocampal slices with the agonist cx614 led to increased dendritic protein synthesis, mediated by bdnf secretion and trkb receptor activation . Additionally, the novel ampa receptor potentiator ly392098 has been shown to increase the expression of bdnf in primary neuronal cultures . Several mechanisms have been proposed to explain the modulation of bdnf induced by ketamine, as schematized in figure 1 . Ketamine - mediated suppression of resting nmda receptor activity can lead to inhibition of eukaryotic elongation factor 2 (eef2) kinase and subsequently to a dephosphorylation of eef2, with a concomitant augmentation of bdnf synthesis . Alternatively, or additionally, the resultant depolarization from ampa receptor activity can activate voltage - dependant calcium channels, allowing calcium influx and exocytosis of bdnf, which can then activate trkb receptors, in turn setting off an intracellular signaling cascade that includes phosphorylation and thus activation of akt (also known as protein kinase b) and extracellular signal - regulated protein kinase (erk). Both akt and erk are involved in the regulation of synaptic protein synthesis (described in detail in the next section). Of note, it has also been proposed that the upregulation in bdnf and synaptic protein expression could be due to the de - suppression of translation and not to activity - dependent bdnf release or intracellular trkb signaling . As the production of synaptic proteins that are important for neuroplasticity is one of the potentially critical steps for ketamine action, attention has recently turned to those enzymes involved in their synthesis . One of these enzymes is the serine / threonine kinase mtor, whose activation is essential in regulating the expression of several proteins involved in synaptic plasticity . Interestingly, reduced synaptic proteins in conjunction with reduced mtor signaling have been found in the pfc of depressed subjects, highlighting the importance of mtor . Therefore, it is worth describing briefly the signaling cascades that both activate and are activated by it (shown in figure 1). Mtor can be phosphorylated by several kinases, including akt and erk, which are both triggered by neurotrophic factor signaling cascades (as described above). Mtor then enables the translation of synaptic protein by activating p70s6 kinase and inhibiting the inhibitory 4e binding proteins (4e - bps). Importantly, administration of ketamine to rats has shown a rapid induction of phosphorylation of mtor, p70s6 kinase and 4e - bp1 in synaptoneurosome pfc preparations . This induction was accompanied by an upregulation of arc, glur1, psd95 and synapsin i, which are all markers of synaptic plasticity and found to be decreased upon exposure to stress in the learned helplessness paradigm of depression . The importance of mtor is emphasized by results obtained on pre - treatment with the inhibitor rapamycin, which blocks cell - cycle progression and prevents p70s6 kinase activation . This inhibition completely repressed the behavioral antidepressant effects of ketamine when tested in both the forced swim test and learned helplessness paradigms . In line with this behavioral effect, rapamycin blocked ketamine induction of layer v pyramidal pfc neuron spine number and function, as well as the expression of synaptic proteins . Furthermore, co - treatment with ketamine and the ampa receptor antagonist, nbqx, completely block 4e - pb1, p70s6k, mtor, erk and akt phosphorylation . A recent study in female rats showed no changes in the phosphorylation of either of them as mediating the response to ketamine, suggesting that at least some of the underlying mechanisms may be sex - specific . It is generally agreed, though, that ketamine - induced synaptogenesis appears to be a result of nmda receptor blockade at rest, which leads to the de - suppression of translation of rapid dendritic proteins and bdnf . Of relevance here, bdnf - evoked protein translation in neuronal dendrites has been reported to be attenuated by both rapamycin and small interfering rnas specific for mtor . It is important to mention that mtor can form two complexes complex 1 (mtorc1) and complex 2 (mtorc2), defined by the presence of regulatory - associated protein of mtor or rapamycin - independent companion of mtor, respectively . Although publications examining the effects of ketamine on synaptogenesis have not yet differentiated between these two complexes, observations with the inhibitor rapamycin would suggest that studies have indeed focused on mtorc1 . It is theoretically possible, though, that mtorc2 is also involved in the action of ketamine as it activates akt and has downstream effects that are important in the organization of the actin cytoskeleton . However, the above - mentioned ability of a brief pre - treatment with rapamycin to abolish the action of ketamine strongly suggests that mtorc1 is the principal effector, given that rapamycin robustly and rapidly inhibits mtorc1 and only partially and slowly acts on mtorc2 . High levels of inflammation have been reported to be important in depression and appear to influence treatment response . We will describe two main mechanisms that have been proposed for ketamine: a direct action on inflammatory cytokines and regulators and an involvement in the kynurenine pathway . A recent meta - analysis has shown that ketamine administration before or during surgery significantly inhibits the early post - operative interleukin (il)-6 inflammatory response in patients . Additionally, ketamine is able to suppress lipopolysaccharide (lps)-induced tumor necrosis factor (tnf)-, il-6 and il-8 production in the human whole blood . Similar results were observed upon ketamine treatment of rats, showing an attenuation of both the increase in tnf- as well as the increase in the ratio of il-6 to il-10 following an escherichia coli endotoxin challenge . Further tests in animals have shown suppression of il-6 and tnf-, as well as nitric oxide, subsequent to an lps insult . A series of experiments have shed light into some of the mechanisms underlying these changes in inflammatory markers . For example, studies in a human monocytic cell line indicated that the immunoinhibitory effects of ketamine appear to be caused by inhibition of activation of the transcription factor nuclear factor - kappa b (nf-b). Interestingly, ketamine has also shown to cause inhibition of the expression of the toll - like receptor (tlr) 4, as well as attenuation of the phosphorylation of p65, one of the subunits of nf-b, in astrocytes challenged by lps . In addition, ketamine - induced inhibition of lipoteichoic acid - induced tnf- and il-6 was also shown to be mediated by inhibition of translocation and transactivation of nf-b . Additionally, ketamine has displayed inhibition of inflammation via upregulation of the inducible heme oxygenase-1, which can provide cellular protection by exerting antioxidative effects ., ketamine has proven to be effective in reducing suicidal symptoms, and recent evidence points toward a low grade of inflammation in the brain of suicide victims . In particular, significantly elevated levels of quinolinic acid (quin), associated with higher levels of il-6, have been reported in the cerebrospinal fluid of suicide attempters . This increase in quin, an nmda receptor agonist, correlated with the scores on the suicide intent scale . Quin is an end product of tryptophan metabolism, in which the enzyme indoleamine 2,3-dioxygenase (ido), induced by cytokines, directs tryptophan away from serotonin and toward kynurenine . Further metabolism of kynurenine can then lead to quin and also, or alternatively, to kynurenic acid (kyna), an nmda receptor antagonist . The increased quin / kyna ratio supports the hypothesis of an overall nmda receptor stimulation, suggesting that changes in glutamatergic neurotransmission could be specifically linked to suicidality . Further support for the participation of the kynurenine pathway comes from a recent study in mice . Exposure to ketamine immediately before or after administration of lps abrogated the development of lps - induced depressive - like behavior (known to occur via activation of ido), without altering the lps - induced sickness . The role of nmda receptor antagonism by ketamine was additionally confirmed when mice pre - treated with the ampa receptor antagonist nbqx displayed a restoration of the depressive - like phenotype upon exposure to both lps and ketamine . A complete evaluation of the regulation of all enzymes within the kynurenine pathway upon ketamine administration will be clearly of great interest . Administration of ketamine to mice has been shown to inhibit brain gsk-3, a kinase that, interestingly, is also a target of mood - stabilizing agents . The inhibition observed upon ketamine administration occurs via an increase in serine - phosphorylation of both the and isoforms of the enzyme . Indeed, animals with a knock - in mutation that blocks gsk-3 phosphorylation did not respond to ketamine treatment in the learned helplessness paradigm, demonstrating that ketamine - induced phosphorylation of gsk-3 is required for its antidepressant properties . Gsk-3 is, in fact, involved in the same pathway as mtor, being phosphorylated and therefore inactivated by both akt and p70s6k, as seen in figure 1 . Furthermore, inhibition of gsk-3 by either short interference rna or through pharmacological agents has also been shown to increase bdnf, thus implicating gsk-3 in synaptogenesis . For example, the gsk-3 inhibitor sb216763 was not able to produce a long - lasting antidepressant action in mice subjected to a chronic mild stress paradigm, when compared with ketamine administration ketamine, through a mechanism that involves this kinase, is able to influence the circadian molecular machinery . Conversely, therapies like sleep deprivation, which are capable of phase - shifting behavioral and physiological rhythms, have been shown to induce rapid improvement in subsets of depressed patients . In particular, it has been shown that acute exposure of animal neuroblastoma cells to ketamine led to a reduction in the amplitude of circadian transcription of the genes brain and muscle aryl hydrocarbon receptor nuclear translocator - like 1 (bmal1), period 2 (per2) and cryptochrom 1 . Furthermore, ketamine altered the recruitment of the circadian locomotor output cycles kaput (clock):bmal1 complex on circadian promoters . Interestingly, the ketamine - induced repression of clock: bmal1 was reduced after treatment with the gsk-3 inhibitor sb216763 . Of note, mtor, mentioned above as being required for the antidepressant effects of ketamine, has been implicated in having a key role in the entrainment of the suprachiasmatic nucleus (the central regulator of circadian rhythms in mammals) to light . Mtor is also involved in the resetting of the circadian clock by regulating the synthesis of the core circadian gene protein, per1 and per2 . Interestingly, high doses of ketamine have been shown to block light - induced phase shifts in locomotor activity when administered to hamsters, further suggesting that circadian rhythm regulation could have an important role underlying the mode of action of the drug . Here we have described evidence supporting the notion that ketamine exerts antidepressant properties mainly via modulation of synaptogenesis and inflammation (and possibly gsk-3). In particular, ketamine is able to improve synaptogenesis by acting on nmda and ampa receptors, and probably through stimulation of mtor activity, which in turn triggers the translation of synaptic proteins required for neuronal plasticity . Additionally, ketamine potentiates bdnf / trkb signaling . Moreover, ketamine is able to reduce inflammation, an effect that could occur via inhibition of nf-b or by preventing ido activation and the possible concomitant shift of tryptophan metabolism toward neurodegenerative metabolites . Finally, gsk-3 appears to have an important role, as its inhibition is required for ketamine to convey its antidepressant effects . Of interest, recent evidence has shown that the antidepressant effects of ketamine were completely abolished when female rats were ovariectomized, and restored upon oestrogen and progesterone supplementation, suggesting a critical role for gonadal hormones . This may be clinically relevant, as ketamine has already shown sex - specific differences, both in rat models of analgesia and catalepsy and in human studies looking at amnestic effects . The growing understanding of the mode of action of ketamine has triggered an increased interest by pharmaceutical companies in the development of novel and more effective antidepressant drugs . Indeed, traxoprodil, an nr2b subtype selective nmda antagonist, and glyx-13, an nmda receptor glycine - site functional partial agonist, are some of those . Interestingly, glyx-13 is currently in a phase ii clinical development programme for treatment - resistant depression . Given the evidence that ketamine is effective in cases of suicidal ideation, improved agents will clearly help to deal with this medical emergency . The hope that a new type of fast acting antidepressants would bring to the growing numbers of mdd patients and their worried families clearly warrants further funding and increased efforts from the scientific community and pharmaceutical companies.
Guillian barre syndrome (gbs) is associated in 45 - 75% of cases with cranial nerve involvement . Facial nerve is the commonest to be involved followed by extra ocular muscles and lower cranial nerve involvement . Only two cases has been reported till date with gbs with total paresis of motor cranial nerves . Here a thirteen year old boy presented with acute progressive areflexic flaccid quadriparesis associated with motor cranial nerve involvement with bilateral facial and bulbar weakness . He had an upper respiratory infection one week preceding the motor weakness which started from the lower limbs . On day seven after the onset of motor weakness of limbs, the child developed significant bulbar weakness, difficulty in talking and could not move the tongue . He was totally anarthric . On day nine, he had significant respiratory muscle weakness requiring mechanical ventilatory support . On day fourteen, the nerve conduction studies (ncs) were suggestive of severe demyelinating motor sensory polyradiculoneuropathy . His anti - ganglioside antibody panel in the blood showed positive igm gm2, gt1b, igg gm1,2,3 and gt1b antibodies . His csf showed albuminocytological dissociation and his antinuclear antibody was negative . His magnetic resonance image (mri) he was treated with intravenous immunoglobulin at a daily dose of 0.4 g / kg for five days . He showed gradual improvement and started swallowing after eight weeks of the onset of illness . The wasting of the tongue also improved gradually at 3 months after the onset of illness [figure 2]. At 6 months repeat ncs was suggestive of motor sensory demyelinating radiculoneuropathy with improvement in conduction velocities and compound muscle action potential amplitudes compared to baseline study . Tongue wasting noted at 2 weeks after the onset of motor weakness improvement in tongue weakness and wasting noted at 6 months twelfth nerve involvement, either isolated or as a part of multiple cranial nerve involvement is quite uncommon and only two cases has been reported till date as a part of multiple motor cranial nerve involvement in a case of gbs ., reported a case of fulminant gbs with quadriplegia and total paresis of motor cranial nerves and polo et al ., reported a case of atypical gbs with multiple cranial neuropathies including xii cranial nerve involvement twelfth nerve involvement, either isolated or as a part of multiple cranial nerve involvement is quite uncommon and only two cases has been reported till date as a part of multiple motor cranial nerve involvement in a case of gbs ., reported a case of fulminant gbs with quadriplegia and total paresis of motor cranial nerves and polo et al ., reported a case of atypical gbs with multiple cranial neuropathies including xii cranial nerve involvement gbs with multiple cranial nerve involvement is a known entity but the involvement of xii cranial nerve is extremely rare . Our patient had multiple cranial neuropathies with involvement of vii, ix, x and xii nerves . He had a severe form of gbs with areflexic quadriparesis along with respiratory muscle involvement requiring prolonged ventilatory support . His mri brain was normal supporting that the tongue weakness is due to xii cranial nerve involvement . Twelfth nerve involvement, either isolated or as a part of multiple cranial nerve involvement is quite uncommon and only two cases has been reported till date as a part of multiple motor cranial nerve involvement in a case of gbs . Tan et al ., reported a fulminant case of gbs with quadriparesis with all motor cranial nerve involvement and their ncs showed segmental demyelination which was responsible for such severe involvement and possibly the pathophysiology in this case and our case are similar . Described a 23 years gentleman who developed a progressive illness over ten days with diplopia, facial diplegia and a nasal voice . Subsequently, the patient also developed weakness of the neck and tongue muscles, dysphagia, abolition of reflexes of the left arm and right triceps but without involvement of the respiratory muscles or other limbs . However, the same patient had involvement of reflexes of left arm and right triceps only without involvement of respiratory muscles and lower limbs . Hence it was thought to an atypical variant of gbs in contrast to our case where he had all the typical features of gbs . Our patient showed anti - gm2 antibody positive which is usually seen in post cmv infection with or without gbs . Usually going on to ventilatory support requirement and the same is found in our patient . The two previous case reports did not have the autoantibody levels to corroborate the clinical and electrophysiological findings . The cranial neuropathy in our patient showed gradual recovery and tongue atrophy recovered over next three months . At 6 months gbs with multiple cranial nerve involvement can rarely involve the xii nerve as well causing significant tongue weakness and such a presentation may be indicative of a severe nature of the disease and it usually recovers over a period of time.
Third molar impaction is the most commonly observed tooth impaction in modern communities, as the third molars are the last teeth to erupt . Third molars have been reported to account for 18 - 32% of impactions . While most studies have reported no gender differences in caucasians, the purpose of this study was to investigate the prevalence, distribution, position, and depth of impacted third molars (itms) in turkish orthodontic patients from a single academic institution . We retrospectively reviewed the panoramic radiographs, intraoral photographs, and dental casts of 207 patients (62 men and 145 women; age, 20 - 39 years; mean age, 22.7 3.29 years) who had undergone orthodontic treatment . Patients with conditions such as cleidocranial dysplasia, and down's syndrome were excluded from the study . If an itm was present, its angle and depth of impaction were recorded . If the third molar was not fully erupted to its normal functional position or the eruption process was not complete with regard to angular position or lack of space, then it was deemed as impacted . The angulation of impaction was measured with reference to the angle formed between the intersected longitudinal axes of the second and third molars . The following classification system was adopted: 0-10, vertical; 11-79, mesioangular or distoangular; 80-100, horizontal, and the remaining cases were classified as cases of inverted or buccolingual impaction . The level of impaction was considered in relation to alveolar bone and the cementoenamel junction of the itm: level a, not buried in bone; level b, partially buried in bone (if any part of the cementoenamel junction was lower than the bone level, the tooth was considered to be partially buried in bone); and level c, completely buried in bone . Statistical analysis was performed using ncss (number cruncher statistical system) 2007 statistical software (utah, usa). Groups were compared using pearson's chi - square test and fisher's exact test . In our orthodontic patient population, itms were evident in 112 patients (54.1%). Prevalence of itm was not found to be significantly different between men and women (61.3%, n = 38 vs. 51.0%, n = 74, respectively), (p = 0.23). The proportion of itms was approximately equally represented in the maxilla and the mandible for both genders . Of the 300 itms, there was no significant difference between the distribution of maxillary and mandibular itms in men and women (p = 0.97). In addition, there was no significant difference in the frequency of third molar impaction between the right and left sides within each arch (p> 0.05). The distribution of itms on the right and left sides was equal (50%) in the mandible . In the maxilla, the distribution was 51.35% for the right side and 48.65% for the left side . Distribution of itms by arch and gender the distribution of patients by number of itms is shown in table 2 . The most common type was impaction of all 4 third molars and 2 third molars . There were no significant differences between the distribution of the number of itms in male and female patients (p> 0.05). Distribution of patients by total number of itms the occurrence of the different angulations of impaction in the mandible is shown in table 3, and their occurrence in the maxilla is shown in table 4 . The most common angulations were mesioangular (65.1%) and horizontal (25.7%) in the mandible, while they were distoangular (64.2%) and vertical (23.6%) in the maxilla . The distribution of the mesioangular and horizontal angulations of impaction in the mandible were significantly different between the men and women (p <0.05) [table 3]. In the mandible, the mesioangular position was more common among women than among men, while this association was vice versa with respect to horizontal position . On the other hand, there was no significant difference in the frequency of different angulation types of impaction between the sexes in the maxilla . Distribution of angulations of mandibular itms distribution of angulations of maxillary itms the distribution of impaction levels is shown in table 5 there were no significant differences between level b and level c impactions in the maxilla (p = 0.75), or in the mandible (p = 0.79) in men and women . The relative proportion of the different levels of impaction was significantly different between the 2 arches . There was significantly more level c impaction in the maxilla (46%) than in the mandible (32%) (p = 0.02). There was no significant difference between level b and level c impactions in the maxilla or the mandible, in men (p = 0.45), or women (p = 0.21). Distribution of levels of impacted third molars in the maxilla and mandible in our patient population, 86 patients presented with bilateral impaction (76.8% of all subjects with itms) [table 6]. The frequencies of maxillary and mandibular bilateral impaction were similar (30.2%, and 24.4%, respectively). Seventy - one percent of the mandibular bilateral impaction cases and 82% of the maxillary bilateral impaction cases presented with the same angle classification and the same level of impaction [table 7]. Bilateral impaction with the same angulation (82%) was significantly more frequent than bilateral impaction with different angulation (30%) at the same level of impaction in the maxilla (p = 0.00). There was no significant relationship between the impaction levels with regard to mandibular bilateral impactions (p = 0.12). There was also no significant relationship between the bilateral impaction angulations in the maxilla and the mandible (p = 0.38). Distribution of bilateral impaction by arch distribution of bilateral impaction by angulation and level in the maxilla and mandible to achieve more reliable results in this study, 20 years was deemed to be the lower age limit on the basis of literature regarding the growth and eruption time of the third molars . In addition, due to possible angulation changes of third molars even after 30 years of age, the upper age limit was 39 in our patient population . Although all of the patient records and file information were carefully investigated, it is still possible that some third molars may have been extracted . For determination of the angular position of an itm, this useful system has also been used before by other researchers . In many other studies, the angulation of itms was usually determined using visual impression based on winter's classification . Due to ethnic variations, differences in diet, and genetic heredity, variations in jaw - tooth sizes and facial growth can occur . Thus, some differences are evident in the prevalence of itms in studies of different populations . In addition, differences in diagnostic criteria, sample sizes, and statistical methods may also lead to differences in results . In our study, many other studies have reported much lower frequencies of itms . In another turkish population study, it was concluded that the prevalence of itms was 35.9%, and this proportion was lower than was observed in our study . On the other hand, saglam and tuzum reported the frequency of lower third molar impaction to be 42.4% and that of upper third molar impaction to be 40.5% in a sample of turkish patients aged between 16 and 75 . There were some methodological differences between these studies with regard to factors such as age limits . Some other prevalence studies have also reported higher itm frequencies, between 65.6% and 76.0%, in american, chinese, indian, and swedish populations . We observed no significant gender differences with regard to the frequency of third molar impactions (p = 0.23). However, some other studies, including another investigating a turkish orthodontic patient population, have reported a significantly greater frequency of itms in women (p <0.05). In our study, the proportions of impacted mandibular (50.7%) and maxillary (49.3%) third molars were almost equal . In contrast, in most other studies, itms were observed more frequently in the mandible than in the maxilla, although some studies have also indicated the opposite . Because of the different classification systems used in different studies, including classification determined solely by visual impression alone, it is difficult to make reliable comparisons of the reported itm angulations . In this study, we found that mesioangular impaction of mandibular third molar and distoangular impaction of maxillary third molar were the most common (65.1%, and 64.2%, respectively). Most other researchers have also reported that mesioangular inclination was the most common, in the mandible . However, hugoson and kugelberg reported vertical impaction to be the most common (50.0%) in the mandible . In their study, in which they used a different classification system, celikoglu et al . Reported that in a turkish population vertical impaction was the most common in the maxilla (58.9%). In our research, study result, and they used the same criteria in their study (level b, 80%). However, they included all third molars, impacted or otherwise, in their study . Thus, our result is not directly comparable to that reported by hugoson and kugelberg, or other study results . In this study, we only evaluated impacted third molars, and our reference was the amount of crown buried in bone . The frequency of level a impaction was reported as only 5% by quek et al . In our research however, level a was observed in only 1 patient and thus was not analysed statistically . There was significantly more level c impaction in the maxilla (46%) than in the mandible (32%) (p = 0.02) and this result is comparable with that of quek et al . Dachi and howel have indicated that the prevalence of unilateral and bilateral impaction of third molars was almost the same . Have reported that bilateral occurrence of third molar impaction was more common than unilateral impactions . Our study also showed that bilateral impaction of itms was more frequent . In this study however, quek et al . Indicated that the majority of bilateral third molar impactions were in the mandible . They also reported that half of the bilateral impactions presented with the same classification of angle and level of impaction in the mandible, while this percentage was 71% in our research . Our study showed that third molar impaction was present in 54.1% of a group of turkish orthodontic patients aged between 20 and 39 years, with no significant gender differences, and this frequency is the highest, thus far, to be reported in a turkish population . Mesioangular and distoangular inclinations were the most common in the mandible and the maxilla, respectively . Of all itms,
Hyperparathyroidism (hpt) occurs when there is an abnormal increase in parathyroid hormone (pth) production by the parathyroid gland [13]. Primary hpt (phpt) is caused by parathyroid adenomas in 85% of cases, leading to hyperplasia and over - secretion of pth . In most individuals in western society, it is diagnosed at an asymptomatic stage, without signs or symptoms of (pth) calcium excess . Initial symptoms are nonspecific, such as weakness, malaise, fatigue and possible mood disturbances . If not diagnosed and left untreated, it leads to devastating consequences including nephrolithiasis, nephrocalcinosis, renal failure, osteopenia and osteoporosis . A 22-year - old male student presented to our hospital with a 2-year history of progressive lower extremity weakness . One year prior to the admission, he had been seen at another medical clinic because of fatigue and difficulty walking . His radiographs showed no fractures, and he was referred to physiotherapy for muscle strengthening . Further questioning revealed that, despite physiotherapy, his weakness had progressed and he had to quit school as he was having considerable difficulty walking up a flight of stairs . A thorough investigation was performed and showed a serum calcium level of 3.46 mmol / l (normal: 2.12.6 mmol / l) and a pth level of 138 pmol / l (normal: 1.69.3 pmol / l). He was referred to our general surgery service where he was admitted for excision of the parathyroid adenoma . Upon admission, one day prior to the scheduled parathyroidectomy, he had a simple, low - energy fall while he was walking on his own to the hospital . Radiographs revealed a displaced subcapital fracture of his left hip, a segmental fracture of his right humerus and extreme osteopenia (fig . 1). Figure 1:(a) left femoral neck displaced subcapital fracture and (b) right humeral shaft segmental fracture . (a) left femoral neck displaced subcapital fracture and (b) right humeral shaft segmental fracture . One day after sustaining this injury, the patient underwent an uncemented left total hip arthroplasty (tha) (fig . 2), and acetabular fixation was supplemented with multiple screws, and he maintained touch - down weight bearing for 6 weeks postoperatively . Postoperatively, the pth level had decreased to 4.9 pmol / l, and the calcium level had returned to normal at 2.42 mmol / l . The calcium level continued to fall, and 2 weeks after the parathyroidectomy, it had reached 1.70 mmol / l . After a further 20 days, it started to normalize and then continued to remain stable, as shown in fig . 3 . Figure 2:(a) left thr and (b) right humerus in a brace . (a) left thr and (b) right humerus in a brace . A graph for calcium level from admission to 7 weeks after surgery we report this case to illustrate the importance of early detection and diagnosis of phpt, which was possible in this young and presumed healthy individual if proper assessment and management were done . Furthermore, sending him for physiotherapy without a clear diagnosis increased the likelihood of missing the diagnosis and progression of the disease . Solitary parathyroid adenoma is the most common cause of phpt, representing 8590% of cases . Symptomatic phpt with skeletal, renal, abdominal and neuro - psychiatric manifestations has become exceedingly rare in developed countries . Our patient was unusual in that he was not diagnosed early and presented with skeletal manifestations resulting in multiple fractures including femoral neck fracture (fnf), which was treated with total hip replacement (thr). At this young age, thr would affect his lifestyle and put him at risk of revision surgery early in his life . The literature clearly shows that the earlier hpt is detected, the more reversible the disease . We believe that, despite the young age of our patient, thr was still the preferred treatment in this situation owing to his poor bone quality, which could have led to failure of fixation; in addition, severe fracture displacement carries a high risk of femoral head avascular necrosis . Yang et al . Found that salvage tha for failed internal fixation following fnf is a more technically demanding procedure with prolonged operative time and larger amounts of postoperative drainage (within 24 h) and that patients are at increased risk of developing hip complications compared with primary thr for acute displaced fnf . French et al . Reported a case of a 21-year - old male with parathyroid adenoma who had presented with a left femoral shaft fracture and right fnfs . Although these were treated with fixations, the right side required conversion to thr within a year of his presentation.
However a permanent type 1 diabetes - like status can be experimentally induced either by total pancreatectomy or by chemical destruction of pancreatic -cells with streptozotocin (stz) [1, 2]. Both experimental approaches allow for the establishment of chronic hyperglycemia and low endogenous insulin production in nhps, similarly to what it is found in humans with type1 diabetes . In patients an optimal treatment of diabetes involves control of glycemia by insulin administrations under haemoglobin a1c (hba1c) monitoring . Daily glucose measurements, even if frequent, do not provide accurate measures of long - term average blood glucose concentrations . The best method to assess long - term glycemic control is the measurement of hba1c . Hba1c values are important parameters for physicians and quite helpful to adjust the dose of insulin and antidiabetic drugs for a better control of the disease . Evidence supporting the translation of hba1c into glycemic control and long - term risk assessment of microvascular complications has been provided by the diabetes control and complications trial (dcct) and the united kingdom prospective diabetes study (upkds). The dcct and the ukpds are landmark clinical trials that compared the effect of intensive glucose - lowering therapies with conventional blood glucose control on the long - term risks of complications in patients with type 1 (dcct) and type 2 (ukpds) diabetes . Both of the trials documented that better glycemic control was associated with improved clinical outcome . Hba1c values are strongly correlated with blood glucose levels and with the risk of developing complications . This is the reason why we thought it useful to record hba1c as a parameter to monitor diabetes in nhps (particularly in long - term islet graft recipients) as in humans . Even if species differences should be taken into consideration, testing of novel therapeutic approaches in nhps is one of the best ways to predict possible effects in humans . Clinical signs vary, but there is often a gradual progression of the disease even in nhps . Traditional tests for the detection of diabetes mellitus include measurement of fasting plasma glucose concentrations, measurement of urine glucose concentrations, oral (ogtt) and iv (ivgtt) glucose tolerance tests, measurement of urine ketone concentrations, and measurement of fasting plasma insulin concentrations [711]. Diagnostic criteria are ideally based on the risk of developing long - term microvascular complications [1114]. While nhps are the recipients of choice for testing alternative sources of pancreatic islets, such tests present challenges in these animals . These include difficulty in sample collection, necessity for anesthesia during blood drawing with the potential for drug interactions, multiple confounding factors (e.g., activity, duration of food withholding, or diet), stress hyperglycemia attributable to restraint (i.e., catecholamine release suppressing insulin secretion), and lack of established reference ranges [1517]. Objectives of the study reported here were to identify values for measurement of hba1c percentage in blood samples obtained from nhps (macaca fascicularis) to determine whether these percentages varied with respect to glycemic control after diabetes induction and insulin treatment . Hba1c measurements were also carried out after pig islet transplantation in diabetic nhp recipients in an attempt to assess whether this physiologic variable can be considered a suitable test to monitor glucose metabolism and provide a positive feedback after islet transplantation, particularly in long - term survivors . Even if islet xenotransplantation of porcine islets in nhps restores normal blood glucose levels in diabetic recipients, to date no clear long - term effect has been fully demonstrated . Glycated hemoglobin percentage can offer a reliable means to determine the establishment of euglycemia after xenotransplantation . A total of 15 male cynomolgus monkeys (i.e., macaca fascicularis, three spring scientific, perkaise, pa, usa), 24 years old and 2.84.9 kg (median 3.4 kg), were included in this study; 6 monkeys were nondiabetic, 9 diabetic, and 3 diabetics received islet transplantations . Gt - dko pigs (-1,3-galactosyltransferase double ko pigs) or hcd46 transgenic pigs (revivicor, blacksburg, va, usa) were used as sources of pancreata for islet transplantation . All animal care procedures were in accordance with the institutional principles of laboratory animal care (national society for medical research) and the guide for the care and use of laboratory animals and were approved by the university of pittsburgh animal care and use committee . Diabetes was inducted in 9 monkeys with 125150 mg / kg i.v . Of zanosar streptozotocin (sicor pharmaceutics, irvine, ca, usa) in a single dose . Diabetes was confirmed by persistent hyperglycemia (> 11.1 mmol / l on at least two consecutive readings) and by the need for insulin to prevent ketosis . Ivgtts (intravenous glucose tolerance test) and asts (arginine stimulation test) were performed 731 days (median 12 days) after induction of diabetes . Infusion of insulin (humulin r; eli lilly, indianapolis, in, usa) to maintain the blood glucose level <11.1 mmol / l and to prevent the development of ketosis . Blood glucose (mmol / l) was measured in whole blood with a portable glucometer (freestyle; abbott laboratories, abbott park, il, usa). Serum levels of primate c - peptide (nmol / l) were measured by radioimmunoassay (linco research, st charles, mo, usa) using species - specific antibodies . Aprotinin 0.05 kiu / l (trasylol; bayer pharmaceuticals, west haven, ct, usa) was added to the serum as a stabilizing agent . To document the metabolic status of each monkey before induction of diabetes, after induction of diabetes, and after islet transplantation until euthanasia, we recorded mean blood glucose (mmol / l), the prevalence of blood glucose readings> 11.1 mmol / l (%), the mean exogenous insulin requirement (iu kg day), and mean porcine (graft) c - peptide levels . Blood samples for hba1c testing were collected fasting every 24 weeks before and after induction of diabetes . Immediately after collection of a sample, blood was transferred into a tube containing edta, which was immediately used for measurement of hba1c percentages . For the measurement of specific hba1c, an inhibition of latex agglutination assay is used (hba1c - specific mouse monoclonal antibody adsorbent onto latex particles, dca vantage analyzer, siemens healthcare diagnostics, deerfield, il, usa). An agglutinator (synthetic polymer containing multiple copies of the immunoreactive portion of hba1c) causes agglutination of latex coated with hba1c specific mouse monoclonal antibody . This agglutination reaction causes increased scattering of light, which is measured as an increased absorbance at 531 nm . Hba1c in whole blood specimens competes for the limited number of antibody - latex binding sites causing an inhibition of agglutination and decreased scattering of light . The decreased scattering is measured as a decrease absorbance at 531 nm . The hba1c concentration the percent hba1c in the sample is then calculated as follows:% hba1c = [hba1c]/[total hemoglobin] 100 . After pancreatectomy in the anesthetized donor pig, islet isolation was carried out according to a modification of the method described for human islets, optimized for pigs that involved low enzyme concentration, low digestion temperature, and minimal mechanical digestion . Intraportal injection of islets (an average of 40,000100,000 islet equivalents / kg body weight) was carried out under general anesthesia of recipients . Continuous insulin infusion was restored if blood glucose was consistently> 11.1 mmol / l . After induction with antithymocyte globulin, immunosuppression was maintained with humanized anti - cd154 monoclonal antibody (abi 793, novartis pharma, basel, switzerland) and mycophenolate mofetil (roche, nutley, nj, usa). Anticoagulation / antiaggregation / anti - inflammatory treatment was achieved with heparin or dextran sulfate, prostacyclin (glaxosmithkline, research triangle park, nc, usa) and aspirin; islet graft function was monitored by measuring porcine c - peptide . A commercially available technical computing program was used for graphic analyses (graphpad prism 4 for macintosh graphpad software, la jolla, ca, usa). Suggested criteria for diabetic classification of subjects were derived from other studies [7, 15] that involved the use of nhps . A commercially available statistical program was used for statistical analyses (graphpad prism 4 for macintosh, graphpad software). Human data obtained from the literature are presented as the range of values or mean of the published data [20, 21]. The data for healthy monkeys were compared with the human data from the literature to better characterize similarities and differences, even though the comparisons are limited by the difference in the testing conditions . For a better characterization of differences between monkeys and humans table 1 also reports fasting blood glucose and c - peptide levels comparisons, based on previous reports . Mmol / l and was significantly lower than the corresponding values in humans (3.95.6 mmol / l; p <.05). Human c - peptide values were consistently lower than monkey c - peptide levels (0.47 furthermore, hba1c values for nondiabetic healthy monkeys were lower than those in humans, with statistically significant difference (4.4 0.1% in monkeys versus 4.99 0.1% in humans, p <.05). The hba1c of nondiabetic monkeys was compared to that of monkeys that were streptozotocin (stz) induced, hyperglycemic, and insulin independent . The increase in hba1c levels following diabetes induction confirms also in nhps the notion that chronically high blood glucose affects glycation of hemoglobin (figure 1(a), p <.05). As expected, during the ivgtt the peak of glucose concentration in diabetic monkeys was significantly higher (p <.05, 2 min after glucose i.v . Thereafter, the glucose levels decreased at a slower rate in diabetic than nondiabetic animals, as shown by the lower kg (mean 1.44 0.2 mmol min, p <.001). The c - peptide increase, seen in nondiabetic monkeys, was absent in diabetic monkeys (figure 1(c)). During the ast in nondiabetic monkeys (figures 1(d) and 1(e)), blood glucose remained stable while c - peptide values rose at 2 min and then returned at prestimulus value at 5 minutes . Published data show that human acrarg is similar to that in monkeys; however, the absolute basal and stimulated values are lower in humans than in monkeys (2223). In diabetic monkeys during the ast, blood glucose remained stable at approximately 10.8 mmol / l, and c - peptide showed no response (arcarg0.400.31 nmol / l). In summary, after stz treatment, blood glucose levels in monkeys increased above 10 mmol / l, and fasting levels of endogenous c - peptide declined to values corresponding to 1218% of the c - peptide levels before diabetes induction . Any residual endogenous c - peptide did not respond to physiological stimuli, as shown by the results of the dynamic tests and by the absence of a correlation between endogenous c - peptide and blood glucose levels . Figure 2 compares the blood glucose profile and hba1c levels in two nhps, during their followup after stz and (in one case) islet transplantation . Figure 2(a) shows the blood glucose levels trend after stz of monkey m12408 . Despite iv insulin treatment blood glucose level tended to increase, and coherently hba1c values (red dots) also tended to increase (from a basal pre - stz value of 4.3% to 8.2% at the end of followup). Figure 2(b) shows a second monkey (m123 - 08) blood glucose profile and hba1c values prior to and after islet transplantation . Whereas blood glucose control and hba1c increased after stz, as also shown in monkey m124 - 08, a substantial decrease in blood glucose levels and a drop in the hba1c level from 9.9% to 6.5 . Better physiological glycemic control was achieved by the islet transplant and was reflected in the improved hba1c values, testifying to a better management of the disease . In this experiment a total of three monkeys were transplanted with porcine islets in the liver via the portal vein; following the transplant, all of them experienced improved metabolic control of glucose . Improvement was defined by a statistically significant drop in between blood glucose levels (8.87 0.19 mmol / l versus 5.28 0.06 mmol / l, p <.0001) and insulin dose (1.07 0.06 iu kg day versus 0.04 0.01 iu kg day, p <.0001). Paralleling these parameters, a statistically significant decrease in hba1c percentage (8.11 0.36 versus 6.16 0.21, p <.0001) was found before and after islet transplantation (figures 3(a), 3(b), 3(c)). Figure 4 shows a summary of the - hba1c values comparing nondiabetic, diabetic, and islet - transplanted nhps . There is a statistically significant difference in hba1c levels between nondiabetic and diabetic nhps and between nondiabetic and islet - transplanted nhps (p <.0001), showing a trend of improvement of hba1c values that parallels the improvement of blood glucose levels, as expected . However, caution is needed in interpreting these results because of the short time between samples . Glycated hemoglobin (hb) is produced when a carbohydrate, such as glucose, binds to an hb molecule, such as hba0, and undergoes intermolecular transformation to form a stable glycated ketoamine product . Hba1c is formed via a posttranslational nonenzymatic attachment of glucose to hemoglobin at a rate dependent on the ambient blood glucose during the lifespan of the red blood cells (approximately 120 days in humans). Glycated hb percentage represents therefore the integrated value for average blood glucose concentrations in the preceding 6 to 8 weeks . A number of different glycated hb forms have been identified, reflecting various attached sugar residues . These forms include hba1a (fructose-1,6-diphosphate, or glucose-6-phosphate), hba1b (ketamine - linked pyruvic acid), and hba1c (glucose). This last form is the most commonly tested, constituting approximately 80% of hemoglobin a1 (hba1). This hemoglobin is a derivate of adult hemoglobin (hba), with monosaccharide (fructose or glucose) attachments . In strict chemical terms, the molecular structure of hba1c is n-(1-deoxy)-fructosyl - hemoglobin or n-(1-deoxyfroctose-1-yl) hemoglobin beta chain . Methods routinely used for the measurement of glycated hb percentage separate the molecule on the basis of its charge, structure, or antigenic properties . The most popular methods rely on the increased negative charge found in the glycated hb molecule to distinguish it from its nonglycated form . These assays include electrophoresis and ion - exchange chromatography as by high - performance liquid chromatography (hplc). Alternatively, boronate affinity chromatography separates glycated hb on the basis of its structure rather than its charge . In this assay, separation is the result of carbohydrate moieties on the glycated hb molecule binding by condensation to the affinity reagent, di - hydroxyboronate . This method is specific for all glycated hbs, irrespective of molecular charge or the site of glycation on the hb molecule . In addition, it is also able to detect the glycated portion of hb in patients with hb variants such as hb s, c, or f. thus, the term total glycated hb has been used to describe this type of assay . Finally, immunoassays, such as the method we have utilized in this study, have been developed for the measurement of hba1c . Monoclonal or polyclonal antibodies generated against the 6- to 8-amino acid peptide of the glycated amino terminus of the -globin chain are here utilized . Advantages to measure hba1c percentage with this technology involve the simplicity of the approach; the need for one small (only few microliters) blood sample; the fact that the assay is relatively unaffected by recent food intake, activity, illness, or stress; it still reflects glycemic control during the preceding few weeks allowing measurement of hba1c even when pathological hemoglobin variants or nhp hemoglobins are involved . Unfortunately, there are also a lot of different variables associated with measurement of glycated hb percentage that could potentially modify those values; for example, processes that decrease the mean lifespan of rbcs will reduce the availability of hb for glycation and, therefore, the percentage of glycated hb, irrespective of glucose concentrations . Similarly, conditions that increase the lifespan of rbcs (e.g., iron deficiency) will increase the amount of glycated hb . Appropriate validation is therefore required for experimental animal models such as diabetic nhp recipients of pig islet xenotransplantations . Reliable measurement of glycated hb percentages would be then useful in determining the efficacy of therapeutic treatments . Glycated hb percentages have been measured, only to a limited degree, in non diabetic, borderline - diabetic, and confirmed - diabetic nhps [17, 2426]. Percentages of hba1 and hba1c in nondiabetic baboons (papio anubis), measured by use of cation - exchange chromatography, are approximately half of those found in humans . It has been suggested that this may be explained on the basis of differences in the life - span of rbcs in humans (100 to 120 days) compared to baboons (30 to 60 days). At least 2 studies [25, 26] have documented an increase in hba1c percentages in borderline - diabetic or confirmed - diabetic nhps . In one group of celebes crested macaques (macaca nigra), the hba1c value increased from 2.6% in nondiabetic macaques to 7.9% in diabetic macaques, as measured by use of electrophoresis . In addition macaques without overt hyperglycemia, but with impaired glucose clearance, impaired insulin secretion, and increased postprandial glucose concentrations, had a significant increase in hba1c content to 3.5% . Reference ranges for glycated hb percentages have not been determined in many nhps, but values that increase by 10% were generally considered to be abnormal . Although the number of animals that were evaluated in our study is small, also reflecting the complexity of the experimental model, we had the opportunity to use the same technology to measure variations in hba1c values within the same animals under different clinical metabolic conditions: prior to diabetes induction, as insulin - dependent diabetics, and after islet transplantation . The levels of hba1c obtained reflected different glucose metabolic conditions such as diabetes, and improved metabolic control following islet transplantation . These values coherently represent the expected trends relative to the experimental metabolic conditions and further validate the technical approach proposed . By using this simple method in broader scale studies and employing larger numbers of animals, a better correlation between hba1c levels and blood glucose levels in cynomolgus monkeys in this study we presented the hba1c values of nondiabetic and diabetic nhps (macaca fascicularis), analyzed with an inhibition of latex agglutination assay (hba1c - specific mouse monoclonal antibody adsorbent onto latex particles). These values are lower than the ones we find in humans without diabetes and very similar to those in diabetic patients under insulin treatment, respectively . Total hba1c percentage can be measured with a user friendly assay and provides useful information for control of diabetes mellitus in nphs, as is done in human patients, and it could be used during the standard management of glucose metabolism in those animals . Our data provide evidence that hba1c can be used to monitor diabetes in streptozotocin - diabetic monkeys and to receive feedbacks on the functionality of xenogenic islet transplantion, particularly useful during long - term followups.
Hemodynamic monitoring and maintaining the heartbeat and blood pressure of patients undergoing spinal surgery is of prime importance . It leads to better control of bleeding in the surgical field, particularly in congested, small and limited areas such as spine . Considering the nature of the spinal fusion surgery as a major surgery with a possibility of bleeding and the fact that this surgery is performed on the elderly or traumatic patients in many circumstances, the goal is to choose a drug with appropriate hemodynamic effects while providing good anesthetic depth and short recovery state . Using a potent and short acting opioid such as remifentanil has been used successfully as an analgesic - sedative drug in the recent years . We chose remifentanil because it is an ultra - rapid opiod with safe and convenient tapering effects . On the other hand, another sedative - hypnotic - analgesic drug with an acceptable effect is dexmedetomidine (dex). Dex has been known as a highly selective 2-adrenoreceptor agonist and has been used as a sedative agent in some operative and clinical situations (1 - 3). Some investigators reported that dex could reduce the propofol requirement in remifentanil - based anesthesia for faster postoperative recovery and more stable intraoperative hemodynamics (4, 5). However, the exact propofol sparing impact of dex during remifentanil - based anesthesia has not been well investigated . Previous studies have found that dex had complex vasodilative and vasoconstrictive hemodynamic effects on pre and postsynaptic 2 -receptors . The effect of dex in a lower dose was vasodilation and its vasoconstriction effects have been presented in higher doses . Reduction of blood pressure and heart rate decreased after long - term dex usage (6,7). Controlling hemodynamic state of patients who have spinal operation is very important, and maintaining their heart rate and blood pressure in normal or low- normal levels can reduce their bleeding loss . To increase anesthesia depth and minimize serious side effects, this study was designed to compare the effect of dex as an analgesic and sedative drug with remifentanil as an adjuvant drug in anesthesia regiment in spinal operation . In the present randomized clinical trial with a parallel design, 60 patients in the age range of 15 - 65 years who were candidates for posterior spinal fusion operation were included . This study was approved in the research ethical committee of iran university of medical sciences; written informed consent was obtained from all the participants . The clinical trial was registered in the iranian clinical trial registry system as irct2012081410336n3 . A pilot study of pain reported by patients after spinal fusion surgery revealed that the average vas score for pain was 3 with a standard deviation of 1.4 . The sample - size calculation was based on a maximum allowable difference of 1 in vas scores . Inclusion of 30 patients in each group provided a power of 0.80 when alpha was set at 0.05 . Samples were gathered using random sampling method and sample size was calculated based on the following formula: considering alpha= 0.05, beta= 20% and the calculation power 80%; the sample size was 60 patients . Eligibility criteria for the patients included in the trial were: patients with asa class i or ii, candidates for posterior spinal fusion surgery in maximum 3 levels, surgical time between 2 - 5 hours, participation agreement, and without cardiovascular, pulmonary, neurological, nephrology and coagulopathy abnormality in their history and physical examination . All the operations were performed in rasool- akram hospital complex affiliated to iran university of medical sciences . Patients were randomized using block randomization method and were equally divided into two trial groups with 30 patients . The monitoring device was massimo (saadat company, iran) and we also used electrocardiography, pulsoxymetry, nibp and capnograph for these patients . Induction of anesthesia was performed for all the patients using fentanyl 3 microgram/ kilogram, midazolam 0.1 milligram / kilogram as premedication and was continued with 0.2 milligram / kilogram cis - atracurium and sodium thiopental 5 milligram / kilogram . This study was double blinded and patients and their surgeons were both blind to the intervention and an unknown person of research team prepared encrypted codes separately . Both groups received isoflurane 1% during the surgery as a maintenance regiment . It has been observed that dex can decrease the heart rate and induce bradycardia in special doses; propofol has this effect as well . So we chose isoflurane which does not have this effect and was a suitable inhalation anesthetic agent for this study . As an intervention in one group, remifentanil (ultiva, abbott, ca) was injected firstly using infusion pump with a dose of 1 microgram / kilogram in 15 minutes and then injected with continuous infusion with a dose of 0.2 microgram / kilogram / min . In the other group, patients received dex (precedex, hospira, usa) firstly via infusion pump with a dose of 1 microgram / kilogram in 15 minutes continued followed by an infusion of 0.5 microgram / kilogram / hour . Dex was prepared as 4 microgram / milliliters in 0.9% sodium chloride infusion . For the postoperative pain control, an infusion pump containing 1000 micrograms of fentanyl was used in the first day followed by a rate of four milliliters per hour . As trial outcomes, heart rate and mean arterial blood pressure (map) was measured before and after induction and every 15 minutes during the surgery . In patients with bradycardia (heart rate<40 bits / minute) and map<50 mmhg, five milligrams of ephedrine was injected and recorded; and in non - responded cases, patients were excluded from the trial . In patients with increased or decreased blood pressure, 10% increase or decrease in isoflurane dosage was used . If blood pressure or heart rate were still elevated, fentanyl 50 microgram was used . In hypertensive patients who were non - responder to isoflourane or fentanyl, nitroglycerine infusion was used and they were then excluded from the study . At the end of the surgery, all the drugs were discontinued and patients were extubated and transferred to the recovery room . In order to provide a similar condition, 50 microgram of fentanyl was injected for patients at the end of operation just after anesthetic drug cessation . Awakening time after anesthetic drugs cessation, time of discharge from recovery, surgeon satisfaction score from surgical field (good, moderate, poor) and bleeding loss were recorded . Pain score, sedation score and the need to analgesic therapy were recorded in first 30, 60,120 and 360 minutes after entrance to the recovery room; so patients were evaluated in the ward . In case of analgesic need (vas>4) in the recovery and ward, 15 mg intravenous ketorolac quantitative variables were presented as mean standard deviation, and qualitative variables were presented as count and percentages . Independent sample t - test and chi - square were used for statistical analysis to compare numerical and categorical data, respectively between the two groups if they had a normal distribution . Repeated - measures analysis of variance (anova) was used to review the results at different time points . In the present randomized clinical trial with a parallel design, 60 patients in the age range of 15 - 65 years who were candidates for posterior spinal fusion operation were included . This study was approved in the research ethical committee of iran university of medical sciences; written informed consent was obtained from all the participants . The clinical trial was registered in the iranian clinical trial registry system as irct2012081410336n3 . A pilot study of pain reported by patients after spinal fusion surgery revealed that the average vas score for pain was 3 with a standard deviation of 1.4 . The sample - size calculation was based on a maximum allowable difference of 1 in vas scores . Inclusion of 30 patients in each group provided a power of 0.80 when alpha was set at 0.05 . Samples were gathered using random sampling method and sample size was calculated based on the following formula: considering alpha= 0.05, beta= 20% and the calculation power 80%; the sample size was 60 patients . Eligibility criteria for the patients included in the trial were: patients with asa class i or ii, candidates for posterior spinal fusion surgery in maximum 3 levels, surgical time between 2 - 5 hours, participation agreement, and without cardiovascular, pulmonary, neurological, nephrology and coagulopathy abnormality in their history and physical examination . All the operations were performed in rasool- akram hospital complex affiliated to iran university of medical sciences . Patients were randomized using block randomization method and were equally divided into two trial groups with 30 patients . The monitoring device was massimo (saadat company, iran) and we also used electrocardiography, pulsoxymetry, nibp and capnograph for these patients . Induction of anesthesia was performed for all the patients using fentanyl 3 microgram/ kilogram, midazolam 0.1 milligram / kilogram as premedication and was continued with 0.2 milligram / kilogram cis - atracurium and sodium thiopental 5 milligram / kilogram . This study was double blinded and patients and their surgeons were both blind to the intervention and an unknown person of research team prepared encrypted codes separately . Both groups received isoflurane 1% during the surgery as a maintenance regiment . It has been observed that dex can decrease the heart rate and induce bradycardia in special doses; propofol has this effect as well . So we chose isoflurane which does not have this effect and was a suitable inhalation anesthetic agent for this study . As an intervention in one group, remifentanil (ultiva, abbott, ca) was injected firstly using infusion pump with a dose of 1 microgram / kilogram in 15 minutes and then injected with continuous infusion with a dose of 0.2 microgram / kilogram / min . In the other group, patients received dex (precedex, hospira, usa) firstly via infusion pump with a dose of 1 microgram / kilogram in 15 minutes continued followed by an infusion of 0.5 microgram / kilogram / hour . Dex was prepared as 4 microgram / milliliters in 0.9% sodium chloride infusion . For the postoperative pain control, an infusion pump containing 1000 micrograms of fentanyl was used in the first day followed by a rate of four milliliters per hour . As trial outcomes, heart rate and mean arterial blood pressure (map) was measured before and after induction and every 15 minutes during the surgery . In patients with bradycardia (heart rate<40 bits / minute) and map<50 mmhg, five milligrams of ephedrine was injected and recorded; and in non - responded cases, patients were excluded from the trial . In patients with increased or decreased blood pressure, 10% increase or decrease in isoflurane dosage was used . If blood pressure or heart rate were still elevated, fentanyl 50 microgram was used . In hypertensive patients who were non - responder to isoflourane or fentanyl, nitroglycerine infusion was used and they were then excluded from the study . At the end of the surgery, all the drugs were discontinued and patients were extubated and transferred to the recovery room . In order to provide a similar condition, 50 microgram of fentanyl was injected for patients at the end of operation just after anesthetic drug cessation . Awakening time after anesthetic drugs cessation, time of discharge from recovery, surgeon satisfaction score from surgical field (good, moderate, poor) and bleeding loss were recorded . Pain score, sedation score and the need to analgesic therapy were recorded in first 30, 60,120 and 360 minutes after entrance to the recovery room; so patients were evaluated in the ward . In case of analgesic need (vas>4) in the recovery and ward, 15 mg intravenous ketorolac quantitative variables were presented as mean standard deviation, and qualitative variables were presented as count and percentages . Independent sample t - test and chi - square were used for statistical analysis to compare numerical and categorical data, respectively between the two groups if they had a normal distribution . Repeated - measures analysis of variance (anova) was used to review the results at different time points . In this study, baseline variables such as age, gender, body mass index, having diabetes mellitus and hypertension were significantly different between the two trial groups . Female / male ratio in the remifentanil group was 9/21, and it was 12/18 (p=0.401) in the dex group . Four patients in the remifentanil group, and 2 patients in the dex group had diabetes (p= 0.890), and 5 patients had hypertension (p<0.001). The mean of blood pressure (p = 0.005) and heart rate (p<0.001) had significant changes in patients who received remifentanil and dex as study intervention compared to the baseline time . In the study patients, dex had a significant lowering impact on intraoperative blood pressure and heart rate compared to remifentanil (p<0.01). Time series of mean arterial pressure (map) and pulse rate (pr) among study participants the mean of sedation scores after extubation in patients who received dex was significantly higher than sedation scores in patients who received remifentanil (p<0.01). Awakening time in patients of the remifentanil group was significantly lower than patients of dex group (3.1115.75 vs. 4.8932.93; p<0.001). There was a significant difference between the two groups in term of need to fentanyl at 105 minutes during the operation (p= 0.010). In other measured times during operation, the need to fentanyl injection had no significant difference between the patients of the two groups . In the first 30 and 60 minutes of entrance to the recovery room, the need to analgesic therapy in the remifentanil group was significantly higher than the dex group (p<0.05); but there were no significant differences between the two groups in the other measured times in recovery . Discharge time in patients of the remifentanil group was significantly lower than the patients in the dex group (3.3933.25 vs. 5.3648.54; p<0.001). The mean of post - extubation pain score of patients in the remifentanil group was significantly higher than the patients in the dex group (0.42.1 vs. 0.31.8; p=0.03). The mean pain scores one and two hours after entering the recovery ward in was significantly higher in the remifentanil group comparing the dex group (p<0.001). Six hours after extubation, the mean pain score was not significantly different between the two groups (0.42.9 vs. 0.82.7; p=0.2). Surgeons were more satisfied with the observed outcomes in patients of the dex group compared to the patients of the remifentanil group (100% vs. 78.6%; p=0.01). Although in this present study the mean blood pressure (bp) and heart rate (hr) revealed significant difference in both drugs, dex had a significant lowering impact on intra - operative bp and hr compared to remifentanil . Sedation after extubation was significantly higher in patients who received dex compared to patients who received remifentanil . In addition, in the dex group lower need to analgesic was observed in some intra - operative times and more specifically in postoperative period . Some previous studies found relatively similar trial findings and reported that dex can make 30 - 50% reduction in the propofol requirement in concomitant use of dex in adolescent patients and healthy volunteers (1, 2). It seems that the sedative impact of dex in patients is mediated by locus ceruleus in the brain stem . In this region laryngoscope insertion and endotracheal intubation can induce the sympathetic nervous system and cause severe tachycardia, hypertension or arrhythmia (10). Dex is a very effective alpha-2 agonist and has more impact on stabilizing cardiovascular system after intubation and reduces the need to analgesic and sedative drugs preoperatively (11). Some investigators such as segal et al . Reported a decrease in requirement for halothane, and aho et al . Reported a decrease in a requirement for isoflurane up to 90% (12, 13). Moreover, in one study, the dosage of thiopental sodium was significantly decreased after using dex in anesthesia (14). Dex can decline the serum level of catecholamine and norepinephrine against some of the stressful stimuli such as intubation (15). Some other studies reported that dex have complex of vasodilation and vasoconstriction impacts, by activating pre - synaptic 2-receptors on sympathetic and post - synaptic 2 -receptors of the central nervous system medicates vasodilation, and by effects on post - synaptic 2 -receptors on vascular smooth muscle cells can mediate vasoconstriction impacts (16 - 18). Some previous studies reported a biphasic and dose - dependent impact for dex on the blood pressure and heart rate of patients (17, 18). On the other hand, dex increases bp in the short - term usage and decreases bp in patients in the long time usage . Dex in low dosage (plasma concentrations, 0.7 - 1.2 ng / ml) causes reduction in the release of norepinephrine release and an inhibition of sympathetic neurotransmission by activating 2a receptors (6, 8, 19). The high dosage of dex (i.e., plasma concentrations,> 1.9 ng / ml) produces 2b receptor - mediated vasoconstriction (6, 8). Most previous studies confirmed cardiovascular depressive effects of dex and reported an increase in the incidence of hypotension and bradycardia (7, 14, 19). The propofol - sparing effect of dex may be beneficial for the reduction of the propofol dosage and may avoid the adverse effects such as myocardial depression, metabolic acidosis, impaired platelet aggregation and extended recovery caused by prolonged and large - dose administration of propofol (20 - 24). This study had some limitations that should be considered for the future studies in this filed . Firstly, our study was performed in one hospital and with patients with one type of operation . Secondly, some of the disease characters in spinal regions might have effects on their hemodynamic changes . For the future studies, it is recommended to match the study variables and randomly select the participants while excluding patients with previous history of hemodynamic changes . Dexmedetomidine in patients with spinal operation is associated with lower postoperative pain score and intraoperative bleeding; hemodynamic effects are also significantly better in the dex group . The authors would like to thank the neurosurgery operation room staff for their cooperation.
Socio - cultural factors are important in the epidemiology and psychopathology of various psychiatric disorders12 including factitious ones . They have an influence on the pathways to seeking medical help.3 in addition, illness behavior has cultural overtones in arab patients.4 factitious disorders are characterized by physical, and psychological signs or symptoms that are intentionally produced with no external incentives to feign illness.5 these disorders have been reported from various cultures.67 a distinction should be made between factitious disorders and malingering . In the latter, the patient produces symptoms with an obvious goal . In factitious disorders, the motivation to be a patient is vague and obscure . Munchausen's syndrome, in its classic description, is an uncommon subtype of factitious disorder which has received great attention.78 this syndrome is the earliest description of a factitious disorder with predominantly physical signs and symptoms . Although factitious disorders are common among males, recent reviews indicated a preponderance of female patients.9 the probable judgement that a particular symptom is produced intentionally is made both by direct evidence and with the exclusion of other causes of the symptoms . Almost in all reported cases of factitious disorders with physical symptoms, however, many such patients have been described as having underlying masochistic, borderline or dependent personality traits.9 although the prognosis is usually poor, patients who have adequate psychosocial support with less severe personality pathology can do better . In this article, a 45-year - old, illiterate, saudi housewife was referred from a medical ward for psychiatric consultation . During her stay in the medical ward, she was investigated for skin lesions distributed mainly on the abdomen and both thighs, but not on her back or other areas of the body inaccessible to the hand . Her case history revealed that two years prior to her current psychiatric consultation, she was hospitalized for vague abdominal pain, but all investigations were normal . After discharge from the hospital, she consulted a traditional healer who treated her by cauterization . During the following two years, she presented with recurrent skin lesions on the abdomen, arms and both thighs . Her illness had created marital discord but there was no family history of mental illness . The examination of the mental state did not show any major disorder . During her stay in the psychiatric ward for observation, evidence of intentional production of skin lesions on healed sites and on new areas was reported . When confronted with evidence of their factitious nature, she denied doing herself any harm . The judgement that the skin lesions were produced intentionally was made by direct evidence from the staff in the ward and by excluding other causes of these lesions . She was discharged from the psychiatric ward after three months and given a follow - up appointment in the psychiatric clinic but she never showed up . A 39-year - old, single, illiterate and unemployed saudi male presented with dramatic severe abdominal pain . He was vague and inconsistent when questioned in detail about the nature of the pain . When all of the investigations proved negative, he started to complain of chest pain . His past history showed that he had been admitted to different general hospitals in saudi arabia, some of which were far from his hometown . The information received from some of these hospitals showed that repeated medical and surgical consultations, including extensive investigations, did not reveal any physical disorder . In addition, psychiatric assessment showed no evidence of mental disorders either . During his stay in the psychiatric unit, he presented with acute renal pain, hematuria and evidence of self - induced blood tinged stool . On confronting the patient about the factitious nature of his complaints after organic causes had been ruled out, he became angry and discharged himself . Two months later, he presented to the hospital in a deaf - mute state but left the hospital when again confronted . Similarly, he did not benefit from traditional healers whom he had visited many times, on the advice of his relatives . One of the essential clinical features of the above two cases is the intentional production of physical symptoms . The first case is that of factitious dermatosis, the second is of chronic physical symptoms associated with multiple hospitalizations . The first case of dermatitis artefacta was preceded by cauterization from a traditional healer which might have acted as a predisposing factor . The second case is similar in presentation to other classic cases of munchausen's syndrome10 described in several cultures . It is important for clinicians to remember that with factitious disorder there could be some real physical illnesses that need appropriate management.11 the behavior of these two patients has cultural dimensions . To travel, females in the saudi culture need mahram (a person whom they cannot legally marry) who might not always be available.12 this requirement restricts their travel creates an important feature of what was known as wandering type of munchausen's syndrome.13 in females, the type of munchausen's syndrome that is characterized by less severe psychopathology, a higher functioning level and less frequent factitious behavior is classified as non - prototypical.14 this classification seems to be justified as revealed in case 1, though it has recently been criticized8 because the criteria for classification are not applicable in a good number of factitious disorder cases . New modern hospitals are common in saudi arabia . For submissive female and male patients, falling sick this might be incorporated into the psychopathology of factitious disorders in saudi culture . In spite of socio - cultural factors, it is clear that doctors awareness and acceptance of the possibility of factitious disorders is a prerequisite to making the diagnosis . Once factitious disorder is diagnosed, it is important to confront the patient but remain supportive . Confrontation should be carefully planned . In saudi culture, it would be inappropriate to inform the relatives that the patient is feigning the symptoms, since this may precipitate psychotic breakdown of the patient . Interestingly, both patients consulted traditional healers during their factitious behavior . Although consulting traditional healers could be explained partially by the religious background of the saudi culture, it is interesting that in spite of the modern treatment available in saudi arabia, patients including those with factitious disorder, still consult traditional healers with their somatic and psychological symptoms.
Furthermore, the site of lesion could indicate surgical excision to prevent continuous microtraumas [13]. Surgical excision could determine loss of substance due to the dimension of the nevus that could not be easily directly repaired . The foreskin is a good autologous full - thickness skin graft in several conditions . The authors report the use of foreskin as skin graft to repair a loss of substance due to excision of an interdigital nevus of the foot . A four - year - old boy presented a 2 cm 1.5 cm congenital compound nevus entirely covering the plantar surface of the second finger of his left foot (figure 1). Paediatric dermatologist's indication was a radical excision because of the site and the dimension of this melanocytic lesion . Primary closure of the skin defect secondary to radical excision of the lesion was not indicated because of the large loss of substance and the risk of retractive scar . Then we performed circumcision and a radical excision of the nevus (figure 3(a)); foreskin, trimmed in a rectangular shape (figure 2), was sutured into the residual defect (figure 3(b))., the patient has normal use of the foot finger with no evidence of contracture (figure 4). Congenital melanocytic nevus is a frequent condition in childhood (0,21%) [1, 2]. The role of these lesions in increasing incidence of cutaneous melanoma is discussed and the prophylactic removal of all congenital melanocytic nevi is not supported: however, the most congenital melanocytic nevi are removed on preventing criteria . The selective excision of suspicious nevi is indicated when the features of a possible malignancy are faced . These features can include change in size or colour, irregular borders, or development of ulcerations . Other features that can justify excision are site and extension of the lesion, multinodular aspect, and the presence of other risk factors (immunodeficiency, dysplastic nevus syndrome, and xeroderma pigmentosa). Excision of larger lesions require the use of local plasty, free tissue skin graft, or even the prior use of a tissue expander .graft should be harvested from hairless areas where the skin is redundant (groin, volar wrist crease, volar elbow crease, and ulnar side of the hypothenar eminence). Foreskin as a source of skin graft most often been used in urethral reconstruction for congenital or acquired penile defects [5, 6], in burn reconstruction, most commonly for eyelid resurfacing, and in syndactyly repair [9, 10]. Newborn circumcision remains controversial; this procedure has potential medical advantages (decreased risk of cancer of the penis and urinary tract infections) as well as disadvantages and risks (bleeding, infection, meatitis, and scarred phimosis). In italy, neonatal circumcision is not routinely performed; this intervention is electively carried out until three years of age to repair congenital phimosis and at all ages in cases of scarred phimosis, recurrent balanoposthitis, and urinary infections . Therefore foreskin is frequently available as tissue graft in paediatric population . In our case, dimension and site (difficult to control) of melanocytic foreskin was available because the boy was also affected by congenital phimosis, so we did not look for another source of skin graft . The most common problem reported after the use of prepuce as donor skin is hyperpigmentation . In our case, hyperpigmentation was not a contraindication for the use of foreskin as skin graft because the lesion was hidden localizated . Foreskin provides a skin of good elastic quality avoiding secondary retraction with a favourable rate of graft intake . Therefore, this source of graft gives the advantage of the absence of scar prejudice at the donor site.
The hchs / sol is a multicenter epidemiologic study of us hispanic / latino adults living in 4 specific communities in the united states . Participants were examined at 4 field centers affiliated with san diego state university, northwestern university in chicago, albert einstein college of medicine in the bronx area of new york, and the university of miami . Details of the sampling methods and design have been published.8,9 hchs / sol recruited a total of 16 415 participants (9835 women and 6580 men) aged 18 to 74 years at the time of screening from june 2008 to july 2011 . The hchs / sol sampling design ensured significant representation of major us hispanic / latino background groups including mexicans, puerto ricans, cubans, central americans, dominicans, and south americans (by self - reported national origin). Participants were recruited through a stratified multistage area probability sample design from communities surrounding the 4 field centers listed above . Eligibility criteria included hispanic / latino ethnicity, ability to travel to the local field center, english or spanish language proficiency, and no plans to move from the area . There were no other exclusion criteria based upon health status, comorbidities, legal residency, or other factors, making hchs / sol representative of the noninstitutionalized hispanic / latino adult population at these 4 major us communities . Hchs / sol procedures and examination during the baseline visit have been previously described.10,11 information pertaining to demographic characteristics and medical history was obtained using interviewer - administered questionnaires by trained staff . Medication use was assessed by participants bringing all medications and supplements used during the last 4 weeks; these were reviewed and coded by clinic staff . Diabetes was defined based on american diabetes association definition12 using 1 or more of the following criteria: (1) fasting serum glucose 126 mg / dl, (2) oral glucose tolerance test 200 mg / dl, (3) self - reported diabetes, (4) hb a1c 6.5%, or (5) taking antidiabetic medication or insulin . Trained and certified clinic staff obtained blood samples, anthropometric and blood pressure measurements on all hchs / sol participants . Height was measured to the nearest 0.1 cm and weight was measured to the nearest 0.1 kg with the use of a balanced scale . Body mass index was calculated as weight in kilograms divided by height in meters squared . After a 5-minute rest, blood pressure was measured 3 times at 1-minute intervals using an automated oscillometric device with the participant in a seated position . The average of the second and third blood pressure measurements was used for this analysis . Hypertension was defined as a systolic blood pressure of 140 mm hg or higher, diastolic blood pressure of 90 mm hg or higher, or on antihypertensive treatment . Specimens were stored at 20c and shipped weekly to the lipoprotein analytical laboratory at the hchs / sol central lab at the university of minnesota medical center . This laboratory participates in the lipid standardization program of the centers for disease control and prevention . Tc was measured using a cholesterol oxidase enzymatic method and high - density lipoprotein cholesterol with a direct magnesium / dextran sulfate method . Triglyceride levels were measured in edta plasma with the use of tg gb reagent (roche diagnostics) on a centrifugal analyzer . High - density lipoprotein cholesterol was measured with an enzymatic method after precipitation of non high - density lipoprotein cholesterol with heparin and magnesium dextran sulfate . Low - density lipoprotein cholesterol (ldl - c) was calculated using the friedewald equation.13 laboratory measurements of tc and ldl - c were obtained for all participants at baseline . Participants were classified as having prevalent hc if they were currently using of cholesterol - lowering medication or if their ldl - c and/or tc exceeded risk thresholds defined by the national cholesterol education program / adult treatment panel iii (ncep / atp iii),14,15 as ldl - c 130 mg / dl and/or tc 240 mg / dl . Ncep / atp iii defined individuals with normal ldl - c levels as those with <130 mg / dl . Consequently, prevalent hc includes participants who were treated with a lipid - lowering drug and those who qualified for treatment according to above thresholds but were not treated . Awareness of hc was defined as an affirmative response to the question, have you ever been told by a doctor or other healthcare professional that you had high cholesterol? Treatment of hc was defined as a positive response to the question, because of your high cholesterol, are you now taking prescribed medicine? Control of hc was defined as meeting ncep / atp iii guideline levels for tc and ldl - c among those who were hc treated . Socioeconomic position (sep) was assessed using years of educational attainment defined as highest degree or level of school completed (less than high school; completed high school or high school equivalent; and education beyond high school) and household income level, classified into groups (<$20 000; $20 000 to $39 999; $40 000 to $75 000 and> $75 000). Acculturation was defined using multiple proxy indicators, including nativity, duration of residence in the united states, and language preference based on language used for the interviews (english versus spanish). Greater years of residence in the united states and english language preference indicated higher levels of acculturation . We applied survey methods using sampling weights to provide weighted frequencies of descriptive variables and population estimates of hc prevalence rates, as well as hc awareness, treatment, and control rates in the hchs / sol target population . Descriptive characteristics are age - standardized to the census 2010 us population . We compared rates of awareness, treatment, and control of hc by demographic factors, anthropometric measurements, lifestyle factors, and clinical and sociocultural profiles . Scott analysis to explore whether the prevalence of established hc awareness, treatment, and control varied significantly among hispanic / latino groups and across categories of sep and acculturation . To assess whether health insurance status confounds the association of sep on hc awareness, treatment, and control, we constructed logistic regression models controlling for the effect of health insurance . To examine whether interrelations exists between acculturative factors (language, nativity, and length of time in the united states) and hc status, we performed additional sensitivity analysis looking at the association of time in the united states and hc control stratified by language preference and nativity, as well as looking at the inverse association of language and hc awareness stratified by time in the united states and nativity . The above analyses were performed with sas version 9.3 (sas institute inc, cary, nc) and sudaan release 10.0.0 (rti). All analyses were weighted to adjust for sampling probability and nonresponse, to make the estimates applicable to the target population from which the hchs / sol sample was drawn in accordance with guidelines suggested by the hchs / sol steering and data analysis committees . The hchs / sol is a multicenter epidemiologic study of us hispanic / latino adults living in 4 specific communities in the united states . Participants were examined at 4 field centers affiliated with san diego state university, northwestern university in chicago, albert einstein college of medicine in the bronx area of new york, and the university of miami . Details of the sampling methods and design have been published.8,9 hchs / sol recruited a total of 16 415 participants (9835 women and 6580 men) aged 18 to 74 years at the time of screening from june 2008 to july 2011 . The hchs / sol sampling design ensured significant representation of major us hispanic / latino background groups including mexicans, puerto ricans, cubans, central americans, dominicans, and south americans (by self - reported national origin). Participants were recruited through a stratified multistage area probability sample design from communities surrounding the 4 field centers listed above . Eligibility criteria included hispanic / latino ethnicity, ability to travel to the local field center, english or spanish language proficiency, and no plans to move from the area . There were no other exclusion criteria based upon health status, comorbidities, legal residency, or other factors, making hchs / sol representative of the noninstitutionalized hispanic / latino adult population at these 4 major us communities . Hchs / sol procedures and examination during the baseline visit have been previously described.10,11 information pertaining to demographic characteristics and medical history was obtained using interviewer - administered questionnaires by trained staff . Medication use was assessed by participants bringing all medications and supplements used during the last 4 weeks; these were reviewed and coded by clinic staff . Diabetes was defined based on american diabetes association definition12 using 1 or more of the following criteria: (1) fasting serum glucose 126 mg / dl, (2) oral glucose tolerance test 200 mg / dl, (3) self - reported diabetes, (4) hb a1c 6.5%, or (5) taking antidiabetic medication or insulin . Trained and certified clinic staff obtained blood samples, anthropometric and blood pressure measurements on all hchs / sol participants . Height was measured to the nearest 0.1 cm and weight was measured to the nearest 0.1 kg with the use of a balanced scale . Body mass index was calculated as weight in kilograms divided by height in meters squared . After a 5-minute rest, blood pressure was measured 3 times at 1-minute intervals using an automated oscillometric device with the participant in a seated position . The average of the second and third blood pressure measurements was used for this analysis . Hypertension was defined as a systolic blood pressure of 140 mm hg or higher, diastolic blood pressure of 90 mm hg or higher, or on antihypertensive treatment . Specimens were stored at 20c and shipped weekly to the lipoprotein analytical laboratory at the hchs / sol central lab at the university of minnesota medical center . This laboratory participates in the lipid standardization program of the centers for disease control and prevention . Tc was measured using a cholesterol oxidase enzymatic method and high - density lipoprotein cholesterol with a direct magnesium / dextran sulfate method . Triglyceride levels were measured in edta plasma with the use of tg gb reagent (roche diagnostics) on a centrifugal analyzer . High - density lipoprotein cholesterol was measured with an enzymatic method after precipitation of non high - density lipoprotein cholesterol with heparin and magnesium dextran sulfate . Low - density lipoprotein cholesterol (ldl - c) was calculated using the friedewald equation.13 laboratory measurements of tc and ldl - c were obtained for all participants at baseline . Participants were classified as having prevalent hc if they were currently using of cholesterol - lowering medication or if their ldl - c and/or tc exceeded risk thresholds defined by the national cholesterol education program / adult treatment panel iii (ncep / atp iii),14,15 as ldl - c 130 mg / dl and/or tc 240 mg / dl . Ncep / atp iii defined individuals with normal ldl - c levels as those with <130 mg / dl . Consequently, prevalent hc includes participants who were treated with a lipid - lowering drug and those who qualified for treatment according to above thresholds but were not treated . Awareness of hc was defined as an affirmative response to the question, have you ever been told by a doctor or other healthcare professional that you had high cholesterol? Treatment of hc was defined as a positive response to the question, because of your high cholesterol, are you now taking prescribed medicine? Control of hc was defined as meeting ncep / atp iii guideline levels for tc and ldl - c among those who were hc treated . Socioeconomic position (sep) was assessed using years of educational attainment defined as highest degree or level of school completed (less than high school; completed high school or high school equivalent; and education beyond high school) and household income level, classified into groups (<$20 000; $20 000 to $39 999; $40 000 to $75 000 and> $75 000). Acculturation was defined using multiple proxy indicators, including nativity, duration of residence in the united states, and language preference based on language used for the interviews (english versus spanish). Greater years of residence in the united states and english language preference indicated higher levels of acculturation . We applied survey methods using sampling weights to provide weighted frequencies of descriptive variables and population estimates of hc prevalence rates, as well as hc awareness, treatment, and control rates in the hchs / sol target population . Descriptive characteristics are age - standardized to the census 2010 us population . We compared rates of awareness, treatment, and control of hc by demographic factors, anthropometric measurements, lifestyle factors, and clinical and sociocultural profiles . Scott analysis to explore whether the prevalence of established hc awareness, treatment, and control varied significantly among hispanic / latino groups and across categories of sep and acculturation . To assess whether health insurance status confounds the association of sep on hc awareness, treatment, and control, we constructed logistic regression models controlling for the effect of health insurance . To examine whether interrelations exists between acculturative factors (language, nativity, and length of time in the united states) and hc status, we performed additional sensitivity analysis looking at the association of time in the united states and hc control stratified by language preference and nativity, as well as looking at the inverse association of language and hc awareness stratified by time in the united states and nativity . The above analyses were performed with sas version 9.3 (sas institute inc, cary, nc) and sudaan release 10.0.0 (rti). All analyses were weighted to adjust for sampling probability and nonresponse, to make the estimates applicable to the target population from which the hchs / sol sample was drawn in accordance with guidelines suggested by the hchs / sol steering and data analysis committees . Of 39 384 individuals who were screened, selected, and met eligibility criteria, 41.7% were enrolled, representing 16 415 persons from 9872 households . Baseline demographics of the hchs / sol population have been previously described.10 in the target population, the prevalence of hypertension was 25%; 40% were obese and 17% had diabetes . Mean levels of tc, non - high - density lipoprotein cholesterol, and ldl - c were 196.0, 145.8, and 120.9 mg / dl, respectively . Forty - eight percent of the population had no health insurance coverage . Among those insured, most were at least partially covered by public health insurance (medicaid and/or medicare). Seventeen percent of those ages 65 years and older were uninsured compared to 45% to 58% of those in younger age groups (table1). There was differential health insurance coverage across hispanic background group ranging from 33% to 80%; it was highest among puerto ricans and lowest among south americans . Acculturation factors also varied, preferential english being highest among puerto ricans and lowest among cubans; time residing in the mainland united states being highest among puerto ricans and lowest among cubans . Health insurance coverage was more common among preferential english speakers; among those participants residing in the mainland us> 10 years and among the us born (table2). Prevalence of high cholesterol * among hispanics according to descriptive characteristics bmi indicates body mass index; chd, coronary heart disease; hchs / sol, hispanic community health study / study of latinos; ldl, low - density lipoprotein; tc, total cholesterol . Defined as ldl 130, tc 240, or on cholesterol medications . N s presented are unweighted counts of total participants in the hchs / sol with respective characteristic . Percentages are weighted row percentages and age - adjusted to a standardized population using 2010 us census . Differential insurance status and measures of acculturation across hispanic background group * n s presented are unweighted counts of total participants in the hispanic community health study / study of latinos; weighted row percentages . The overall prevalence of hc was 45%; 15% had a high tc, 35% had elevated non - high - density lipoprotein cholesterol levels, and 37% had elevated ldl - c levels . Hc was seen in significantly higher proportions among males and those in older age groups . In addition, proportions of those with hc were significantly higher among those with concomitant hypertension, diabetes, and those who were overweight / obese compared to those without . Proportions of those with hc were not significantly different across the hispanic / latino background groups or among those with or without self - reported coronary heart disease (table1). Hc awareness was defined as ever being told by a health professional of having hc . Among those with hc, almost half (49%) were not aware of their condition . Among those with ldl - c 190 mg / dl, only 63% were hc aware . A significantly higher proportion of hispanic / latinos were hc aware in the older age groups . Despite a higher prevalence of hc in men, rates of hc awareness were higher among women than men across all age groups, but this difference was most pronounced among those middle - aged and older (figure1a). Significantly higher proportions of those who have hypertension, higher body mass index, or diabetes were hc aware . Us - born hispanic / latinos had significantly lower rates of being hc aware compared to those who were foreign born . A proportion of those who were hc aware was significantly higher among preferential spanish speakers compared to those who preferred english . The lowest and highest sep categories (by educational attainment or income) had the highest rates of hc awareness . Those with insurance coverage had significantly higher rates of being hc aware compared to those who were insured (table3). Logistic regression models controlling for health insurance to address confounding did not attenuate the effect of sep (either income or education) on hc awareness . Individuals of central american and cuban heritage had the lowest rates of being hc aware, while those of puerto rican and dominican backgrounds were more likely to be aware than other groups (figure2). High cholesterol awareness, treatment, and control rates according to descriptive characteristics * bmi indicates body mass index; hchs / sol, hispanic community health study / study of latinos . N s presented are unweighted counts of total participants in the hchs / sol; percentages are weighted row percentages . Percentage of participants (stratified by age and sex) who were (a) hc aware; (b) hc treated; and (c) hc controlled . Hc indicates high cholesterol, defined as treated with a lipid - lowering drug or those who qualified for treatment according to ldl - c 130 mg / dl and/or tc 240 mg / dl but were not treated; ldl - c, low - density lipoprotein cholesterol; tc, total cholesterol . Treatment of hc was defined as taking prescribed cholesterol medicine . Among those who either had laboratory values indicating hc or had been told by a health professional that they had hc, less than a third (30%) were hc treated . Among those with an ldl - c 190 mg / dl, only 14% were hc treated . Although the likelihood of being hc treated increased with age, only 32% and 54% of middle - aged and older hispanic / latinos, respectively, with hc were actually being treated . A larger proportion (> 90%) of hc - eligible hispanic / latinos a trend was observed where rates of hc treatment were higher among hispanic / latino men versus women in the older age groups, particularly among those 65 + years of age (figure1b). Those with diabetes, the lowest sep (by either education or income), and insurance coverage were more likely to be among those hc treated . Controlling for health insurance did not attenuate the effect of sep on hc treatment . Us - born hispanic / latinos had lower rates of being hc treated than those who were foreign born . Among the foreign born, rates of being hc treated increased with longer duration of us residence (table2). Higher proportions of individuals of dominican or puerto rican background were receiving treatment for hc, whereas hispanic / latinos of mexican or central american background had lower rates of treatment (figure2). Hc control was defined as meeting ncep / atp iii guidelines for tc and ldl - c . Among those with hc using cholesterol - lowering medications, older hispanic / latinos tended to have a higher rate of being hc controlled than those who were younger . This sex difference persisted across all ages but was most pronounced among those 65 years and older (figure1c). The presence of diabetes, higher income levels, and insurance coverage was associated with being hc controlled . Controlling for health insurance did attenuate the effect of sep on hc control . Those with greater duration of us residence had significantly higher rates of hc control than more recent immigrants (table2). Rates of adequate hc control varied significantly across hispanic / latinos background groups ranging from 54% to 71%, with cubans and south americans having the lowest hc control rates, whereas puerto ricans had the highest (figure2). Hc awareness was defined as ever being told by a health professional of having hc . Among those with hc, almost half (49%) were not aware of their condition . Among those with ldl - c 190 mg / dl, only 63% were hc aware . A significantly higher proportion of hispanic / latinos were hc aware in the older age groups . Despite a higher prevalence of hc in men, rates of hc awareness were higher among women than men across all age groups, but this difference was most pronounced among those middle - aged and older (figure1a). Significantly higher proportions of those who have hypertension, higher body mass index, or diabetes were hc aware . Us - born hispanic / latinos had significantly lower rates of being hc aware compared to those who were foreign born . A proportion of those who were hc aware was significantly higher among preferential spanish speakers compared to those who preferred english . The lowest and highest sep categories (by educational attainment or income) had the highest rates of hc awareness . Those with insurance coverage had significantly higher rates of being hc aware compared to those who were insured (table3). Logistic regression models controlling for health insurance to address confounding did not attenuate the effect of sep (either income or education) on hc awareness . Individuals of central american and cuban heritage had the lowest rates of being hc aware, while those of puerto rican and dominican backgrounds were more likely to be aware than other groups (figure2). High cholesterol awareness, treatment, and control rates according to descriptive characteristics * bmi indicates body mass index; hchs / sol, hispanic community health study / study of latinos . N s presented are unweighted counts of total participants in the hchs / sol; percentages are weighted row percentages . Percentage of participants (stratified by age and sex) who were (a) hc aware; (b) hc treated; and (c) hc controlled . Hc indicates high cholesterol, defined as treated with a lipid - lowering drug or those who qualified for treatment according to ldl - c 130 mg / dl and/or tc 240 mg / dl but were not treated; ldl - c, low - density lipoprotein cholesterol; tc, total cholesterol . Treatment of hc was defined as taking prescribed cholesterol medicine . Among those who either had laboratory values indicating hc or had been told by a health professional that they had hc, less than a third (30%) were hc treated . Among those with an ldl - c 190 although the likelihood of being hc treated increased with age, only 32% and 54% of middle - aged and older hispanic / latinos, respectively, with hc were actually being treated . A larger proportion (> 90%) of hc - eligible hispanic / latinos <45 years of age were not receiving hc treatment . A trend was observed where rates of hc treatment were higher among hispanic / latino men versus women in the older age groups, particularly among those 65 + years of age (figure1b). Those with diabetes, the lowest sep (by either education or income), and insurance coverage were more likely to be among those hc treated . Controlling for health insurance did not attenuate the effect of sep on hc treatment . Us - born hispanic / latinos had lower rates of being hc treated than those who were foreign born . Among the foreign born, rates of being hc treated increased with longer duration of us residence (table2). Higher proportions of individuals of dominican or puerto rican background were receiving treatment for hc, whereas hispanic / latinos of mexican or central american background had lower rates of treatment (figure2). Hc control was defined as meeting ncep / atp iii guidelines for tc and ldl - c . Among those with hc using cholesterol - lowering medications, older hispanic / latinos tended to have a higher rate of being hc controlled than those who were younger . This sex difference persisted across all ages but was most pronounced among those 65 years and older (figure1c). The presence of diabetes, higher income levels, and insurance coverage was associated with being hc controlled . Those with greater duration of us residence had significantly higher rates of hc control than more recent immigrants (table2). Rates of adequate hc control varied significantly across hispanic / latinos background groups ranging from 54% to 71%, with cubans and south americans having the lowest hc control rates, whereas puerto ricans had the highest (figure2). To our knowledge, there have been no previous studies of diverse hispanic / latino representation comparing hc awareness, treatment, and control among diverse hispanic / latino background groups . Our findings show that hc is highly prevalent among us hispanic / latino adults and that less than half of those with hc were aware of their condition (ie, reported being told by a doctor or health professional that their cholesterol level was high). Moreover, less than a third of those with hc were being treated . Among those receiving treatment gender disparities were observed in that men had higher hc prevalence rates than women but were less likely to be hc aware . Although hispanic / latino women with hc had similar rates of hc treatment compared to men, women were less likely to be hc controlled . Hispanic / latinos ages 18 to 44 years with hc were more likely to be unaware of having hc and to be untreated for this condition compared to those ages 45 and older . Hc treatment rates and optimal cholesterol goal attainment rates for all hispanic / latino background groups were suboptimal . Our study found that among hispanic / latino adults, prevalence of hc was higher among those with hypertension or diabetes, and the presence of these comorbid conditions increased the likelihood of being hc aware, treated, and controlled . Both diabetes and hypertension are associated with substantially increased risk of developing coronary heart disease.16 thus, it is encouraging that among hispanic / latinos with these comorbid conditions, rates of hc treatment and control were among the highest . The presence of more comorbidities probably increases the likelihood of having received the attention of the healthcare system and being hc treated . However, it creates a gap with regard to primary prevention, making those with hc and fewer comorbidities less likely to be among those hc aware, treated, or controlled . Furthermore, our findings showed that even among hispanic / latino individuals with these comorbid conditions, only slightly more than half of those with diabetes and two thirds of those with hypertension were hc aware . Several studies have examined awareness, treatment, and control of hc in the united states (table4) but most have focused on non - hispanic whites or blacks . The cardiovascular health study18 found that among adults ages 65 to 75 years, hc treatment was 8.1% in men and 10.0% in women in 19951996 . The minnesota heart survey19 showed that among non - hispanic whites in 2002, 55% were hc unaware, and 33% were aware and untreated . The genoa study20 looked at 2 us communities of non - hispanic / latino whites and blacks and found that fewer than 1 in 3 of these adults were hc treated and fewer than half of those treated had achieved recommended goals . Studies that included hispanic / latinos primarily involved mexican heritage participants or considered hispanic / latinos as a single aggregate group . Data from the national health and nutrition examination survey demonstrated substantially lower rates of awareness, diagnosis, treatment, and control among mexican americans compared to non - hispanic whites.6,7 less than half of national health and nutrition examination survey mexican americans had been screened for hc compared to 65.2% and 57.7% of non - hispanic whites and non - hispanic blacks, respectively.6 in the multiethnic study of atherosclerosis,5 hispanic participants were significantly less likely to be hc treated and controlled than non - hispanic whites; however, these differences were attenuated by adjustment for healthcare access variables . There is prior data suggesting less intensive cholesterol management in women than men, resulting in a similar disparity in cholesterol control24,25; however data across racial / ethnic groups are somewhat limited . In national health and nutrition examination survey 20052006, women were less likely to be hc controlled than men (56.9% versus 72.1%), and this trend was present across non - hispanic whites, non - hispanic blacks, and mexican americans (although seemingly most disparate among non - hispanic blacks).6 prior studies of high cholesterol awareness, treatment, and control in the united states mesa indicates multi - ethnic study of atherosclerosis; nhanes, national health and nutrition examination survey . Our study illustrates that among hispanic / latinos who are hc treated, the rates of hc control are fairly high (ranging from 54% to 71% among the various hispanic / latino background groups). Studies suggest that gaps in hc treatment and control are often related to gaps in availability of, access to, or continuity of health care.2628 the affordable care act is intended to reduce some of these gaps29 by both increasing insurance coverage among the nonelderly us population and by providing coverage for cholesterol screening with no cost - sharing . However, access to care alone may not resolve gaps in hc treatment and control completely . Other factors that may affect hc treatment and control in the us hispanic / latino population include primordial risks (eg, diet), language barriers, poor patient provider communication, and insufficient treatment (eg, provider clinical inertia) and/or patient medication adherence.30,31 lower out - of - pocket costs, improved patient provider communication, and simplification of the drug regimen generally are associated with better adherence.32 the fact that there is a large gap between hc awareness and subsequent treatment and control underscores the need to go beyond screening programs and health fairs to improve the health of the hispanic / latino population . Those with the lowest sep (by household income), while more likely to be hc aware, had a markedly lower prevalence of hc control . Furthermore, the lowest and highest sep groups (by educational attainment or income) had the highest rates of hc awareness, suggesting a disparity gap among working poor and middle - class hispanic / latinos . It may be that hispanic / latinos in the lowest and highest sep categories are more likely to have health insurance, due to medicaid for those with low sep and more wealth resources for those with higher sep, respectively . Hispanic / latinos in the middle sep group, however, may be more likely to fall through gaps . Higher rates of hc awareness, treatment, and control were related to having health insurance . Controlling for health insurance did not remove the effect of sep on hc awareness and treatment but did remove the effect of sep on hc control . Compared with non - hispanic whites, hispanics are less likely to have health insurance or a regular source of care, and less likely to receive preventive services.33 increased acculturation (by nativity and preferential english speaking) was associated with decreased hc awareness . However, among immigrants, increasing years in the united states did increase the likelihood of hc treatment and control, perhaps due to increasing ability to navigate the us healthcare system and establishment a social support system to do so . Being a more recent immigrant was a more dominant acculturative factor in determining hc awareness than being able to dominate the english language . Similarly, increasing time in the united states portends a greater likelihood of hc control among those who were less acculturated (preferential spanish speakers and those foreign born) but was not a factor among the more acculturated (preferential english speakers and those us born). Health relationship differential effect was also seen in a prior study.34 our findings of variation in hc awareness, treatment, and control across the different background groups provide further evidence of the complexity and heterogeneity of the hispanic / latino population; thus, hc awareness, treatment, or control rates for 1 group cannot be extrapolated to represent all hispanic / latinos . Hispanic / latinos of puerto rican and dominican background seem to fare better with regard to higher rates of hc awareness, treatment, and control whereas those of mexican, cuban, and central and south american backgrounds seem to fare worse . Us hispanic / latinos have differential regional patterns of settlement in the united states, with mexicans concentrated in the southwest, cubans in the southeast, central / south americans in the midwest and south, and puerto ricans and dominicans in the northeast . Thus, some of these differences in hc treatment and control observed among hispanic / latino background groups may be geographical in nature, given differences in medical coverage across state lines . State laws governing medical coverage for low - income individuals vary and these differences are likely to worsen, with some states having declined to adopt the medicaid expansion provisions of the affordable care act . Our results should be interpreted in light of several limitations . While hchs / sol is the largest epidemiologic study of diverse community - dwelling us hispanic / latinos, it was not designed to be nationally representative but is representative of the communities sampled; thus, our findings may not be generalizable to all us hispanic / latinos . Our findings may underestimate the prevalence of hc, since older persons residing in nursing homes or other institutions (with potentially a higher prevalence of age - related hc) were not included in the hchs / sol . Data regarding awareness was based on self - report and could be subject to recall bias . The ncep / atp iii report identifies concentrations of ldl - c rather than tc as the primary target of treatment . However, insights about tc are still of value and participants may not have been able to distinguish between tc and ldl - c when asked whether they had their cholesterol checked and/or had been told by a physician that their cholesterol is high . Our definition of treatment included coding of medications reviewed by study staff; thus, our measure is more robust than that of several prior studies of hc treatment that used medication self - report only . This study was initiated, laboratory samples were collected, and management strategies examined under the older ncep / atp iii guidelines, which focused on target levels rather than atherosclerotic risk to determine who is treated . It is likely that under the new, broader american heart association / american college of cardiology cholesterol treatment guidelines,35 an even larger number of hispanic / latinos will be undertreated, further highlighting this health disparity . In conclusion, we found high rates of hc and a very low prevalence of hc awareness among hispanic / latino adults and a major gap between hc awareness and hc treatment that bodes poorly for the public health of this large and growing segment of the us population . Not surprisingly, our observational data indicated that once treated, achievement of optimal hc control is possible among hispanic / latinos . Complementary and targeted public health programs to raise hc awareness and increase the proportion of hispanic / latinos receiving hc treatment and achieving hc control are needed to reduce healthcare disparities in this population . Our findings fill major gaps in understanding hc awareness, treatment, and control among us hispanic / latino adults, and will help guide and inform physicians and policymakers toward improving patient education programs and disease management . The hispanic community health study / study of latinos was carried out as a collaborative study supported by contracts from the national heart, lung, and blood institute (nhlbi) to the university of north carolina (n01-hc65233), university of miami (n01-hc65234), albert einstein college of medicine (n01-hc65235), northwestern university (n01-hc65236), and san diego state university (n01-hc65237). This study was partially supported by nhlbi grant r01 hl104199 (epidemiologic determinants of cardiac structure and function among hispanic / latinos: carlos j. rodriguez, md, mph principal investigator). Dr rodriguez has received consulting agreement / honorariums from alnylam and amgen research support from the national institutes of health and the american heart association.
Gaucher disease (gd) is a rare autosomal recessive hereditary disorder of glycosphingolipid metabolism, characterized by the accumulation of glucosylceramide in cells of the reticulo - endothelial system, due to deficient activity of the lysosomal enzyme, beta - glucosidase (glucocerebrosidase, ec 3.2.1.45). There are various manifestations of gd but the disease is generally described by three main phenotypes: type 1 gd (gd1), which can manifest at any age and is distinguished by a lack of primary neurological manifestations, and type 2 (acute) and type 3 (chronic) neuronopathic phenotypes which manifest in early life, involve the central nervous system, and result in early death . Usually in infancy (type 2) and childhood or early adulthood (type 3). Type 1 gd is, by far, the most common form of gd . The age of onset, rate of disease progression, severity of symptoms, and the organs affected can vary widely . Some patients with gd1 may remain asymptomatic although the majority experience symptoms that commonly include fatigue, splenomegaly, anemia, thrombocytopenia, hepatomegaly, and skeletal disease . The skeletal manifestations of gd, which may lead to significant pain, morbidity, disability and a progressive reduction in quality of life, include abnormal bone remodeling, osteopenia, osteoporosis, lytic lesions, avascular necrosis, pathological fractures, osteonecrosis of humeral or femoral heads, and vertebral collapse . Osteopenia is a common and often early manifestation of gaucher - related bone disease which may lead to reduced bone mineral density (bmd), reduced peak bone mass, fragility fracture and poor bone healing . Treatment for the systemic manifestations of gd has been available for over 20 years in the form of enzyme replacement therapy, firstly as alglucerase (ceredase; genzyme corporation, cambridge, ma, usa), and subsequently by the recombinant form imiglucerase (cerezyme, genzyme corporation, cambridge, ma, usa). In more recent years, an alternative treatment, substrate reduction therapy, has also become available for adult patients who are unwilling or unable to be treated with enzyme therapy . Another enzyme replacement therapy has also become available: velaglucerase - alfa (shire human genetic therapies). Before disease - specific treatment became available for gaucher disease, splenectomy was frequently carried out to control bleeding risk and the mechanical effects of an enlarged spleen in severely affected patients . It is now known that splenectomy may be associated with an exacerbation of gd - related bone disease . Skeletal involvement is an important indication for treatment to ameliorate symptoms, prevent osteoporosis and reduce risk of pathological fractures . While some skeletal symptoms of gd, such as bone pain and bone crises, may be ameliorated within 12 years by treatment with imiglucerase enzyme replacement therapy, it is recognized that the response of some bone manifestations, such as bmd, to treatment can be slower, and some bone complications, such as avascular necrosis, are irreversible despite optimal treatment . This underlines the need for early initiation of therapy if the risk of irreversible complications is to be reduced . It is known that bmd is accrued mostly in the first 20 years of life, peaks in the third decade, and then declines, suggesting that there is a limited therapeutic window in which to gain maximal benefit from treatment . Similarly, it has been shown that the risk of avascular necrosis (avn) appears to be reduced in patients who initiate enzyme replacement therapy within two years of diagnosis compared with those initiating therapy after two years, although the risk of avn may not be eliminated in patients receiving enzyme therapy . One of the challenges of monitoring bone involvement and treating bone manifestations is that the pathophysiology of bone disease is not completely understood . Although the infiltration of gaucher cells into the bone marrow and bone is thought to be a central event in the pathophysiological process, it is unclear whether, and to what extent, complications are due to mechanical effects, such as compression, and/or the indirect effects of gaucher cells on bone (through the activation of inflammatory mediators), or whether gd alters bone homeostasis independently of gaucher cells . The erlenmeyer flask deformity has been shown to be present in up to 59% of gd patients, suggesting that bone remodeling disorders are common in this disease and may have relevance to skeletal pathologies . Bone turnover or remodeling in healthy individuals is a lifelong process by which mature bone is resorbed (mediated by osteoclasts) and new bone tissue is formed (mediated by osteoblasts followed by calcification). Osteoclasts are recruited from bone marrow precursors which attach to the surface of bone and break down the bone matrix through the release of proteolytic enzymes to leave surface pits . Osteoblasts differentiate from connective tissue precursors and fill the bone pits with a protein matrix which subsequently becomes fully mineralized bone . The biochemical processes involved in bone turnover result in measurable changes in enzyme activities, the release of various bone components, and the expression of proteins involved in the mineralization of the bone matrix . Table 1bone biomarkers used to assess bone turnover.markers of bone formationmarkersourcespecificity for bonespecimencarboxyterminal propeptide of type 1 procollagen (picp)pro - collagenweak (also in soft tissue)serumamino (n-)terminal propeptide of type 1 procollagen (pinp)pro - collagenweak (also in soft tissue)serumosteocalcin (oc)protein produced by mature osteoblasts in bone matrixhigh . The major non collagen protein of bone secreted by osteoblasts . Reflects both bone formation and bone resorption.serumtotal alkaline phosphatase (ap)osteoblast enzymelow: present in plasma membrane of osteoblasts but also liver, kidney, intestinal, and spleen cellsserumbone specific alkaline phosphatase (bsap)osteoblast enzymehighly specific for bone - but up to 20% cross reactivity between bone and liver enzymesserummarkers of bone resorptionpyridinolines (free and total pyr)collagen breakdownspecific (primarily bone and cartilage)urinedeoxy - pyridinolines (d - pyr) (also known as d - pyrilinks, pyrilinks - d, or deoxypyd)collagen breakdownhighly specific (primarily bone and cartilage)urinehydroxyproline (hp)collagen breakdownweak (found in bone, soft tissue, diet)urinecarboxyterminal telopeptide of type 1 collagen (ictp)collagen breakdownhighly specific (primarily bone)serumcross - linked type 1 collagen c - terminal telopeptide (ctx) amino (n-)terminal telopeptide (ntx)collagen breakdownhighly specific (primarily from bone)serum and urinecross - linked type 1 collagen c - terminal telopeptide (ctx) n - terminal telopeptide (ntx)collagen breakdownhighly specific (primarily from bone)serum and urinetartrate - resistant acid phosphatase (trap) (trap5b)osteoclast enzymetrap also found in other tissues specific for osteoclastsserumcathepsin kproteolytic enzymespecific for bone osteoclastsserumnf - kb ligand (rankl) and osteorotegerin (opg)osteoclast recruitment and activityosteoclastsserum the activity of osteoclasts and osteoblasts is normally tightly coupled, but in several pathological states and with increasing age the turnover process may be disrupted . Signaling networks between osteoclasts and osteoblasts are central to the regulation of bone turnover . Osteoclasts, for example, have a role in regulating the activity of other cells, including osteoblast precursors, by participating in immune responses and by secreting cytokines that may affect their own function and those of other cells . Osteoclast formation, in turn, is dependent on the cytokine rankl through binding to its cognate receptor rank on the surface of osteoclast precursors . Osteoprotegerin (opg), a decoy receptor for rankl, inhibits osteoclast differentiation and osteoclast activity . The interaction between opg and rankl, therefore, has a role in regulating osteoclast formation and provides an additional means of estimating bone turnover . The opg / rankl system is of particular interest because it has been exploited as a therapeutic target in some bone diseases . Bone biomarkers have been used to assess disease status and the response to therapy in a number of bone disorders, including post - menopausal osteoporosis, paget's disease, rheumatoid arthritis, and cancer . Here, we examine the literature for evidence of abnormalities in markers of bone turnover in patients with type 1 gd . While biomarkers of activated macrophages, such as chitotriosidase, are often used to assess the whole body burden of gaucher cells and the whole body response to therapy, there is currently no recommended biomarker that is specific to bone . Skeletal disease in gd may develop silently, may not correspond with systemic disease in terms of onset, severity or rate of progression, and may not always be detectable by the commonly used imaging methods . Some bone imaging methods, especially the more sensitive techniques such as quantitative chemical shift imaging (qcsi), are expensive, complex and not widely available for routine monitoring . The identification of a sensitive biomarker specific for bone disease in gd would, therefore, be of significant benefit for detecting and monitoring bone involvement, and would help clinicians determine optimal timing of treatment for the prevention of bone complications . The available english language literature on bone markers in gd was reviewed using online search methods such as pubmed . An overview of biomarkers in gd patients who were untreated, treated with enzyme therapy, and treated with bisphosphonates is to be found in table 3 . Table 2summary of findings on the use of bone biomarkers in gaucher disease.studypatientsstudymarker of bone formationmarkers of bone resorptioncomments / relationship with severity of bone diseaseuntreatedstowens et al . 198526 (subset of 327 cases aged 1865 yrs) all patients had symptoms of skeletal involvement spleen status?retrospective review of untreated gd patient data9 of 26 patient abnormally low urinary hydroxyproline excretion - none excreted high levels gd: 34mg/24hrs (range: 964) (normal 2577 mg/24hrs)concluded that while some patients had reduced bone turnover, the majority had enhanced bone cell activity (measured by kinetic marker tetracycline)drugan et al . 200216 (5 m; 11 f) mean age: 27.44 yrs (954 yrs)bssi scoresstage 2: n=9stage 3: n=4stage 4: n=3spleen status?untreated gd patients compared with 29 age matched controls)oc (3.5 times reduced vs controls: p=<0.003)ctx: 1.9x reduced in gd (3617.62 + /536.69 ng / ml; vs 6808.57 + /865.66 ng / ml in controls: p<0.01)reduced bone turnover and imbalance favouring bone resorption.biomarkers decreased with age in controls; not in gd.no correlation of bone markers with chitotriosidase . 200310 (5 m; 5 f)mean age: 33.2 yrs (range 2540 yrs)bssi scoresstage 12: n=3stage 34: n=4stage 5: n= 2 (1 patient not assessed)6 patients osteopenic (t score 2.5 to 1sd) 3 patients normal bmd4 patients asplenicuntreated patients compared with 10 age and gender matched healthy controlsbsap (normal) gd: 28 u / l; controls 32.06 u / l (p=0.31) oc (normal) picp: reduced in gd (101.17 ng / ml vs 140.75 ng / ml; p=0.0016)ictp: increased in gd (4.24 ng.ml vs 2.87 ng / ml in control; p=0.012) normal parathyroid hormone, ca, p, hydroxyproline, free d - pyrimbalance in bone turnover favouring bone resorption . No statistically different bmd scores between bssi score groupsvan dussen et al . 201140 (17 m; 23 f) median age: 41.8 (range 15.276.2) all patients: bone complications (defined as history of avn, pathological fractures, vertebral collapse, osteomyelitis and bone crises / infarctions). 11 patients asplenic disease severity scored by zimran ssiin 30 patients, samples taken before or within 1 mth of starting treatment . 3 patients received pamidronate results compared with normal reference rangesoc: decreased in gd (median 0.35 nmol / l) pinp (within normal range) median 37 g / l (ref range:1996 g / l)ctx (within normal range)decreased bone formation . Patients with bone complications had significantly lower oc vs patients without complications no significant difference in ctx or pinp between patients . Oc: statistically significant negative correlation with chitotriosidase (cr: 0.323, p=0.042), bmd, and positive correlation with qcsi scores (cr: 0.423; p=0.025) negative correlation between oc and zimran ssi (0.531; p=<0.001)enzyme replacement therapy (ert)gd patients treated with ert vs healthy controlsciana et al . 200512 (6 m; 6 f)mean age: 32.3 yrs(range 2540 yrs)bssi score: stage 12: n=7stage 34: n=4stage 5: n=12 patients asplenicall patients received ert (6.830 u / kg/2wks for 4.5 years) + vitamin d (interrupted in all during first 2yrs of treatment) 12 age and gender matched healthy controlsat baseline: picp: reduced in gd (100.52 ng / ml vs 142.45 ng / ml in controls; p=0.017) bsap, oc, parathyroid hormone, ca, p, hydroxyproline, free deoxypyridinoline normal although reduced urine ca / creatinine in gd group vs controls (ns).ictp: at baseline increased in gd: (3.93 ng / ml vs 2.72 ng / ml in controls; p=0.004after 4.5 yrs, no significant change in bone formation markers -except urine ca / creatinine ratio, (from 0.065 mg / mg at baseline to 0.191 after 4.5 years (p=0.0014)9 of 11 patients (1 not assessed for full period) showed an increase in bmd during follow up (p=0.039)no correlation between bone markers and bmdgd patients treated with ert vs gd patients treated with calcitriolschiffmann et al . Asplenic all had bone involvement at baseline (mri bone score mean=10 (range 611) (score = max number of sites of bone involvement out of 11 sites scored in pelvis and lower extremities)2 year study3 groups:1: calcitriol (control) for 6 mths + ert after 6 mths2 . Calcitriol and ert3 . Ert alone(ert; 60u / kg/2 wks, halved after 1 6 mths in all.in ert treated group: bsap: significant increase (p=0.002) oc: significant increase (p=0.008)in ert treated group: d - pyr: transient increase; returned to baseline after 2 years.calcitriol - no effect on bone density . Ert associated with progressive reduction in bmd - although marked improvement in lumbar spine fat fraction noted . Authors suggest ert may have led to increased bone turnover, with sustained elevation of osteoblastic activity but only transient elevation of osteoclastic activity.gd patients treated with ert over timesims et al . 200833 (19m/14f) 1270 yrs 5 patients asplenic at baseline: skeletal pain (73%); lytic lesions (36%); medullary infraction (36%) osteoarticular necrosis (3%); spine fracture (3%); osteopenia (lumbar spine); (58%) osteoporosis (19%)longitudinal study: all ert treated: most 60u / kg/2 wks (9 dose reductions) bone markers measured every 6 months for 4 yearsat baseline: oc: normalbsap: normalmedian post treatment oc, bsap increased by 60% in first 12 months and remained above baseline (p= 0.050.001)at baseline: ntx: normald - pyr: normal between 36 and 48 months median ntx and d - pyr decreased 2040% (ns).shift towards new bone formation relative to bone resorption after starting treatment reflected in bmd increases over timeert followed consecutively by srtmikosch et al . 20087 (5m;2f) mean age 42.6 yrs (range 2484 yrs) 1 patient asplenic 1 patient vit d deficient 5 with vitamin d insufficiencypatients treated consecutively with ert (1 mth - 7 yrs) and srt (minimum 6 mths). Results compared at 6mths and 1218 mths after change to srtoc: decreased during follow up (ns) ap: decreased significantly during follow upctx: no significant change trap5b: no significant change - remained within normal rangeoc and ctx in lower reference range 1 patient showed intermittent increase in oc and ctx 1 patient showed increasing trap5b - also experienced increasing bone painbisphosphonatesciana et al . 19775 (2 m; 3f) 2460yrs severe skeletal involvement: osteopenia, osteosclerosis, osteonecrosis, severe chronic pain spleen status?pamidronate treatment for 35 mnths (45 mg/3 weeks+ 500 mg calcium daily) in otherwise untreated gd patientsoc: decreased (p=0.04) compared to untreatedhydroxyproline: decreased (p=0.08) total d - pyr: (p=0.08) urinary ca (p=0.04) compared to untreatedbmd increased (p=0.04) compared with baselinewenstrup et al . 200434 (10 m; 24 f) age range: 1850 years 1 year study: 2 arm double blind placebo controlled study for alendronate disodium spleen status?1 yr 2 arm double blind placebo controlled study all received ert (1560u / kg/2 weeks) for at least 24 mths+vitamin d (400u / day)+calcium carbonate (1.5g / day) 1 arm received alendronate (40 mg / day)oc: reduced in alendronate group bsap: reduced in alendronate group (significantly reduced from 6 months 18 months; differences not maintained at 24 months)ntx: reduced in alendronate group d - pyr: no changeat screening all markers in normal reference range (even subjects with more severe osteopenia)significant improvement in bmd in lumber spine in alendronate group.findings do not support a simple osteoclast - mediated mechanism for gd bone manifestationsstudies involving mixed groups (various treatment history / mixed gd type)fiore et al . 2002 (untreated+variable treatment period)12 (8 m; 4 f) mean age: 33 + /13yrs (range 2160yrs) 9 patients asplenic bone involved scored by bssi): 3 patients with severe bone disease (chronic pain, joint replacement / disability)gd patients with variable treatment history (ert: 10 patients at 3060u / kg for 28 yrs) results compared with age and gender matched controls (database)bsap (normal)oc (normal)pyr (increased in urine; pyr=28.63+/ 7.6 nmol / mmol (normal=12.8 25.6 nmol / mmol) d - pyr (increased in urine; d - pyr=6.13+/2.33 (normal 2.35.4 nmol / mmol)increased bone resorption in gd . 2006 (untreated+treated for variable period + some treated with bisphosphonates)173 (74 m; 99 f) mean age: 36.9+/19.9 range: 1856 reported to have mild gd 20 patients asplenic 32 controls108 treated with ert . Many patients (unspecified) also treated with bisphosphonatesopg: (no difference between gd patients and controls)serum opg increased with age in patients and controls . No correlations between opg and gender, disease severity, need for ert, avn, bmd and spleen status . 3 opg polymorphisms detected, but no correlations between polymorphisms and bone density.yoshino et al . 2007 (type 1, and 3 patients, adults and children, variable treatment period)8 (type 1 and type 3 gd); 7 m; 1f; 4 children (no manifest bone disease) 4 adults (evidence of bone disease) 3 patients asplenicexamined role of cytokines in bone disease (compared with published age / gender matched control data) all received ert (1mth11.75yrs)adults: bsap: (normal)oc: (normal)children: bsap marginally elevated in alloc: lower than reference range.adults: ntx: slightly increased in 3 patients (serum); slightly increased in 2 patients (urinary) d - pyr: elevated in 3 patients . Children: ntx elevated in 3 d - pyr lower than reference range in allil-18 (in 7/8 patients) and tgf-1 elevated (all). Concentration of il-18 correlated with bsap in adults, and oc in all 8 patients.gd, gaucher disease; m, male; f, female; oc, osteocalcin; ctx, c - terminal telopeptide; bsap, bone specific alkaline phosphatate; ictp, carboxyterminal telopeptide of type 1 collagen; picp, carboxyterminal propeptide of type 1 collagen; pinp, amino (n-)terminal propeptide of type 1 procollagen; ert, enzyme replacement therapy; srt, substrate reduction therapy. herman bssi (hermann et al . 1997): as detected by x ray imaging . Stage 1: diffuse osteoporosis; stage 2: medullary expansion; stage 3: osteolysis; stage 4: necrosis / sclerosis; stage 5: destruction and collapse (12 mild involvement; 34 moderate involvement; 5: severe involvement) gd, gaucher disease; m, male; f, female; oc, osteocalcin; ctx, c - terminal telopeptide; bsap, bone specific alkaline phosphatate; ictp, carboxyterminal telopeptide of type 1 collagen; picp, carboxyterminal propeptide of type 1 collagen; pinp, amino (n-)terminal propeptide of type 1 procollagen; ert, enzyme replacement therapy; srt, substrate reduction therapy . Herman bssi (hermann et al . 1997): as detected by x ray imaging . Stage 1: diffuse osteoporosis; stage 2: medullary expansion; stage 3: osteolysis; stage 4: necrosis / sclerosis; stage 5: destruction and collapse (12 mild involvement; 34 moderate involvement; 5: severe involvement) table 3overview of biomarker activity in untreated gaucher disease (gd) patients, gd patients treated with enzyme replacement therapy, and gd patients treated with bisphosphonates (excluding studies involving both untreated and treated patients).untreatedmarkernormalincreaseddecreasedformationbsapocpicppinpresorptioncalcium / creatininectxhydroxyprolined - pyrictpntxin response to ertmarkerno changeincreaseddecreasedformationbsapocpicpresorptioncalcium / creatinehydroxyprolineictpin response to bisphosphonatesmarkerno changeincreaseddecreasedformationbsapocresorptiond - pyrhydroxyprolinentx*7 of 12 patients included in the study by ciana et al 200538 were also included in the study by ciana et al . 2003ert, enzyme replacement therapy; bsap, bone specific alkaline phosphatase; ctx, type 1 collagen c - terminal telopeptide; d - pyr, deoxy - pyridinolines; ictp, carboxyterminal telopeptide of type 1 collagen; ntx, amino (n-)terminal telopeptide of type 1 collagen; picp, carboxyterminal propeptide of type 1 procollagen; pinp, amino (n-)terminal propeptide of type 1 procollagen . 7 of 12 patients included in the study by ciana et al 200538 were also included in the study by ciana et al . 2003 ert, enzyme replacement therapy; bsap, bone specific alkaline phosphatase; ctx, type 1 collagen c - terminal telopeptide; d - pyr, deoxy - pyridinolines; ictp, carboxyterminal telopeptide of type 1 collagen; ntx, amino (n-)terminal telopeptide of type 1 collagen; picp, carboxyterminal propeptide of type 1 procollagen; pinp, amino (n-)terminal propeptide of type 1 procollagen . Three studies were carried out in untreated gaucher patients in which results were compared with healthy control data . One study examined bone biomarkers in patients from whom samples were taken before or within one month of starting enzyme therapy, considered here as a study of untreated patients . Of the studies examining bone markers in treatment - nave patients, stowens et al . Found decreased hydroxyproline excretion in 9 of 26 patients (35%) compared with controls, suggesting reduced bone turnover (resorption) in these patients . Reduced bone turnover was also found in a study involving 16 untreated gaucher patients, in whom there was a 3.5 times decrease in osteocalcin levels and a 1.9 times decrease in type 1 collagen c terminal telopeptide (ctx), suggesting reduced osteoblastic and osteoclastic activity but with a net imbalance in bone turnover favoring bone resorption . No correlation was found between bone markers and genotype, chitotriosidase activity, or the zimran severity score index (ssi) for gd . A significant difference was, however, noted in serum osteocalcin levels and ctx according to the stage of skeletal involvement, as assessed by the hermann bone severity score index, with both markers exhibiting the highest levels in stage 3 disease . A reduction in bone biomarker levels in the more advanced stage 4 disease was suggested to be related to an extended loss of functional bone tissue due to osteonecrosis, with a marked decline in both osteoblast and osteoclast activity . A further study involving 10 gd patients also concluded that there was increased bone resorption in gd compared with healthy controls based on a decrease in carboxyterminal propeptide of type i procollagen (picp) and an increase in carboxyterminal telopeptide of type 1 collagen (ictp) while osteocalcin was unchanged . No differences were found in hydroxyproline or free deoxypyridinoline (d - pyr; a more sensitive marker unaffected by collagen synthesis or dietary collagen) or the urinary calcium / creatinine ratio between gaucher patients and controls . There was no correlation between bone marker levels and patient demographics, splenectomy status, spleen and liver volume, or bone mineral density . Found that osteocalcin was decreased in 50% of gd1 patients (n=40) with no significant change in type 1 collagen ctx, a marker of bone resorption, and concluded that imbalances in bone turnover result primarily from a decrease in bone formation . Osteocalcin levels showed a statistically significant negative correlation with chitotriosidase activity, a positive correlation with qcsi scores (0.323, p=0.042; and 0.423, p=0.025, respectively) and other bone turnover markers pinp and ctx (0.483, p=0.002; and 0.466, p=0.002, respectively). Osteocalcin also showed a negative correlation with the zimran ssi score (0.531, p<0.001). The correlations between ssi values and qcsi scores remained significant when the possible confounding contribution of patients receiving bisphosphonates (3 of 40 patients, 7.5%) or who had a recent bone crisis (4 of 40 patients, 10%) were excluded . There was also a negative correlation between osteocalcin and bmd in a subset of patients relatively mildly affected (n=12; r=0.639; p=0.885; p<0.001) but no statistically significant correlation between osteocalcin and the bone marrow burden score (- 0.382; p=0.08). In an analysis of pre - treatment data cited by studies investigating bone marker levels during treatment, sims et al . Found normal biomarker levels [osteocalcin, bone specific alkaline phosphatase (bsap), type 1 collagen n - terminal telopeptide (ntx) and d - pyr] at baseline while ciana et al . One study examined bone biomarkers over time in enzymetreated patients in whom bone markers were measured every six months for four years . Another looked at markers in enzyme - treated patients (who also received interrupted vitamin d supplementation) treated for 4.5 years compared with healthy controls . Compared bone markers in 29 splenectomized patients treated with either enzyme therapy alone or enzyme therapy and calcitriol (1,25-dihydroxyvitamin d3), and mikosch et al . Examined biomarkers in patients treated with enzyme therapy (one month to seven years) followed consecutively by substrate reduction therapy with follow up for up to 18 months . Ciana et al . Found no significant change in bone markers over 4.5 years after the initiation of enzyme replacement therapy except for a statistically significant increase in the calcium / creatinine urinary ratio . Patients showed a gradual improvement in bmd over the 4.5 years which was not reflected in bone biomarker levels . Sims et al . Found that the markers of bone formation (osteocalcin and bsap) increased by approximately 60% in the first 12 months after the initiation of enzyme replacement therapy (without any other medications) and remained above baseline for the remainder of the study (four years). Patients reported a reduction in bone pain and bone crises . At baseline, 13 of 33 patients had a history of bone crises; 2 patients reported bone crises in the first year, one in the second year, and none thereafter . In a study involving splenectomized patients, bsap and osteocalcin increased significantly after the initiation of enzyme therapy while there was a transient increase in d - pyr (marker of bone resorption). Clinically, enzyme therapy was associated with a progressive reduction in bmd in these splenectomized patients, although there was an improvement in lumbar spine fat fraction, an indication of the burden of gaucher cells in the bone marrow . In patients treated consecutively with enzyme replacement therapy and substrate reduction therapy, osteocalcin and alkaline phosphatase were within the normal range (lower part of reference ranges) at the time of the change from enzyme replacement therapy to start of reduction therapy, and decreased during follow up of 1228 months (statistically significant for alkaline phosphatase). There was no statistically significant change in markers of bone resorption (ctx, trap5b). Imaging parameters of bone disease (mri, dexa) generally remained stable throughout follow up, although one patient experienced worsening bone parameters observed by mri . In untreated patients with severe skeletal involvement, pamidronate therapy (plus calcium) resulted in a decrease in osteocalcin, hydroxyproline, total d - pry, and urinary calcium after 35 months of therapy compared to baseline . The second study involved gd patients who had all received enzyme replacement therapy for at least 24 months (plus vitamin d and calcium). Biomarkers were within normal reference range at baseline even in patients with severe osteopenia (t<2). One group of patients received bisphosphonate therapy (alendronate) for 24 months . There was a significant reduction in osteocalcin and bsap for a period of 18 months after the initiation of bisphosphonates but markers of bone resorption were inconsistent with reduced ntxs, but not d - pyr links . There was a statistically significant increase in bmd in the alendronate treated group (p=0.001). Three studies looked at different mixed groups of patients: i) patients who had and those who had not been treated with enzyme therapy (and for varying periods of time); ii) patients who had been treated with other medications such as bisphosphonates; iii) patients who were diagnosed with different gd types (types 1 and 3) and who had been treated with enzyme replacement therapy for varying periods (1 month to over 11 years). Examined bone biomarkers in 12 patients, 10 of whom had received enzyme treatment for 28 years . Markers of bone formation were within normal ranges while the markers of bone resorption, pyr and d - pyr, were significantly increased over control values, leading the authors to conclude that bone resorption was up - regulated in gd . Were the only authors to examine the role of the rankl / opg system in gd - related bone disease . They found no difference in serum opg between gd patients and controls, and did not identify any correlation between opg and disease severity . The authors concluded that opg - related osteoclast activity was probably not a major underlying mechanism for bone disease in their cohort . The patients (n=173) were described as having non - severe involvement and were heterogeneous in terms of age, spleen status and treatment (untreated, enzyme - treated for variable periods, or treated with bisphosphonates). A third study, which mainly focused on examining the role of cytokines in bone remodeling, involved both pediatric and adult patients: 4 with type 1 and 4 with type 3 disease . Osteocalcin and bsap were found to be within normal ranges in adults, while bsap was marginally elevated and osteocalcin was below the normal reference range in children . Studies involving bone markers in gd cover a wide range of different study designs: patient groups in terms of the severity of bone disease, demographics, treatment regimens, and bone biomarkers . Such diversity means it is difficult to make a direct comparison between one study and another . . However, the available evidence suggests variability in whether, and which, biomarkers are elevated or decreased in both untreated gd and treated gd patients (tables 2 and 3). No one bone biomarker appears to reflect the presence of bone disease consistently across multiple studies of gd patients or the response to enzyme replacement therapy . Patients may have differed in the severity of their disease and also in their bone manifestations, both within and between studies . Three pathological presentations may be associated with gd and often all three are presented together: i) focal lytic and/or sclerotic lesions as a result of infarction, thrombosis and inflammation; ii) local disease such as remodeling deformities and cortical bone thinning; and iii) generalized osteopenia and osteoporosis . It is not known whether, and to what extent, different pathological processes in gd - related bone disease might influence bone biomarker profiles, or how recent bone events might have affected overall results . Recent bone crises, for example, may result in increased levels of bone markers since bone crisis is associated with enhanced bone resorption . Similarly, bone markers may increase by 2060% for periods of up to six months following a fracture . Most of the bone markers studied in gd have been used to assess the response to treatment in other disease states, notably osteoporosis . In these other conditions, a decline in bone biomarkers within weeks of starting bisphosphonate therapy indicates that anti - resorptive therapy is having an effect, even in the absence of noticeable changes in bmd, which may take several years . Studies in gd patients that measure biomarker response after the initiation of bisphosphonate therapies showed a decrease in bone biomarkers . A reduction in osteocalcin and hydroxyproline, and osteocalcin, bsap and ntx, together with statistically significant increases in bmd suggest that bone markers may be responsive to bisphosphonate intervention in gd, and that the use of biomarkers may be relevant to the study of bisphosphonate treatment response . The observation that biomarker levels changed significantly in response to bisphosphonates in these studies also underlines the possible confounding effects of attempting to compare biomarkers in bisphosphonate - treated gd patients with controls and correlating biomarkers with clinical parameters in populations that include both bisphosphonate and non - bisphosphonate - treated patients . For example, magel et al . Found no differences in opg levels between gd1 patients, many of whom were treated with bisphosphonates, and healthy controls . In our own studies of the opg / rankl system in 5 enzyme - treated gd patients who had not been exposed to bisphophonates, we found a statistically significant decrease in opg, and a consequent statistically significant decrease in the rankl / opg ratio (p=0.0002) compared with age and gender matched controls . No statistically significant differences in the levels of picp, osteocalcin or trap5b were found (giuffrida et al ., 2010, data not cited). This review does not clarify whether biomarkers could be applied reliably to the measurement of the bone response to enzyme replacement therapy or srt . Markers of bone formation, osteocalcin and bsap increased by 60% in the first 12 months of enzyme treatment in one study with no significant change in markers of bone resorption, suggesting a shift towards new bone formation . Increased bmd has been demonstrated in another long - term study of enzyme - treated gd patients (who also received interrupted vitamin d supplementation). Another study examining biomarker levels in a group of gd patients receiving enzyme therapy, all of whom had undergone splenectomy, demonstrated statistically significant increases in osteocalcin and bsap (p=0.002 and 0.008, respectively) with only a transient increase in d - pyr . However, bmd decreased in enzyme - treated patients despite an apparent shift towards enhanced bone formation, as demonstrated by bone biomarker levels . Splenectomy in gd patients is associated with exacerbated bone disease thought to be related to an increased burden of gaucher cells in bone . However, baseline data in splenectomized patients did not indicate that bsap, osteocalcin and d - pyr were outside the normal reference range . Several other studies also included splenectomized patients in their cohorts . Including the study by sims et al . These data tend to suggest that bone markers may not reflect skeletal changes that might occur as a consequence of splenectomy . Unlike bisphosphonate therapy, where treatment is targeted directly at reducing bone resorption by a process believed to be related to increased osteoclast apoptosis, any effects of enzyme therapy (and srt) on bone biomarkers are likely to be indirect and may occur through different mechanisms . The limited evidence of changes in bone markers in gd appears to suggest that bone markers tend to decrease in response to bisphosphonate therapy (consistent with the response in non - gd populations) but tend to increase in response to enzyme therapy (table 3). Enzyme replacement therapy aims to reduce the burden of glucocerebroside in macrophages and thereby reduce gaucher cell infiltration into organs . Glucocerebroside laden macrophages in bone tissue could have an impact on bone turnover by displacing bone cells, causing vascular occlusion, increasing intra - osseous pressure, and/or by influencing osteoblast and osteoclast activity through inflammatory mechanisms . Several studies have reported disturbances in the levels of serum cytokines in gd, including il-1, il-6 and tnf, which may affect osteoclasts, their function, and bone turnover . Indeed, yoshino et al . Found correlations between il-18 and bsap and osteocalcin, and m - csf and urinary ntx, although in a limited number of enzyme - treated type 1 (n=4) and type 3 gd patients (n=4). A reduction in glucocerebroside burden, therefore, might be expected to impact bone turnover, but the temporal relationship between initiation of therapy and changes in different bone turnover markers has not been studied in detail . The rate of glucocerebroside clearance is likely to depend on multiple factors, such as vascularization, degree of osteonecrosis, fibrosis, and sanctuary sites in the bone in individual patients . The difficulties of determining whether and how biomarkers may be affected in gd, and their response to therapy, are compounded by the knowledge that bone biomarkers fluctuate according to age, diet, exercise, hormonal status, stage of the menstrual cycle, season, the use some medications (e.g. Anticonvulsants, estrogen - based therapies, corticosteroids, insulin, fluoride, vitamin d supplements, calcitonin), immune factors, and inflammatory status, and that different biomarkers show different degrees of spontaneous and intra - individual variability . The response of markers of bone formation and bone resorption may also vary; bone resorption markers are reported to respond faster (212 weeks) than markers of formation (36 months). Urinary biomarkers may also show diurnal variation; pyr and d - pyr are reported to show 2-fold differences over a period of 24 h, and biomarker measurements may show inter - laboratory variation, even when identical assays and methods are used ., statistically significant correlations between biomarker levels and several measures of clinical bone status have been demonstrated in gd in one study . One of the limitations of the studies reviewed here is that bone marker levels are often not the study end point, and data on bone biomarkers were of secondary interest . There have been no detailed studies of changes in biomarker levels over time in untreated and treated gd and their relationship with bone manifestations, leaving clinicians with very little guidance on which biomarkers might be useful in clinical practice . The ideal biomarker in gd should: i) accurately reflect the presence and activity of disease; ii) predict clinically meaningful outcomes; iii) change rapidly with the response to therapy; iv) be easily measured and stable in accessible clinical samples; v) be specific and sensitive; vi) be subject to little or no genetic variation; vii) be reproducible according to agreed standards of measurement; and viii) be inexpensive to measure . Evidence from currently available studies does not suggest that any one bone biomarker, or group of biomarkers, reliably reflects the presence and/or activity of disease or changes in a predictable manner to enzyme replacement therapy . Bone biomarkers may also be subject to considerable variation as a result of patient status (hormonal, medications, etc .) Or as a result of inter - laboratory variability . Bone markers reflect activity in the whole skeleton and may not be specific or sensitive enough to detect subtle localized changes over a general background of activity . In addition, most studies tend to report biomarker levels as being indicative of either bone formation or bone resorption, although some markers may be released as a consequence of both processes . Osteocalcin, for example, is produced by mature osteoblasts in the bone matrix, but is also released from the bone matrix during resorption . There is no evidence as to whether bone markers might have any predictive value in terms of the progression of bone disease or the need for treatment . A greater understanding of the temporal relationship between bone biomarkers and clinical consequence is required . The difficulties of measuring biomarkers are compounded in rare diseases, such as gd, where few patients are available for study and where bone disease may reflect multiple pathological mechanisms . Future advances in identifying meaningful biomarkers of bone disease are, therefore, likely to depend on large internationally coordinated studies . Such studies might be able to compare carefully controlled groups of patients in sufficient numbers for meaningful statistical analyses to be made . Key questions to address include the temporal relationship between fluctuations in bone biomarkers and clinical disease . Do biomarkers measured today reflect current bone abnormalities or do they reflect an ongoing pathological process, the consequences of which are not yet measurable by dexa, mri or other commonly used monitoring methods? Studies involving bone biomarkers in gd show variable results which do not currently support their routine use for the clinical assessment of bone status, as an indication for therapy initiation, or for monitoring the response to therapy . A greater understanding of bone markers and their relation to the bone manifestations of gd is required.
Inhabit the coasts of south korea the yellowtail kingfish (s. lalandi), yellowtail (s. quinqueradiata), and greater amberjack (s. dumerili) (kim et al ., 2001). Yellowtail kingfish do not only occur in south korea, however, but also in temperate waters worldwide, including japan and china (nfrdi, 2004). In japan, 3 species of s. lalandi, s. quinqueradiata and s. dumerili are cultivated (nakada, 2000). Yellowtail kingfish are also cultivated in australia and new zealand (moran et al ., 2007; kolkovski & sakakura, 2007), as well as in california, usa (stuart & drawbridge, 2012). Aquaculture of seriola spp . In south korea has been attempted, beginning with pond culture of yellowtail in 1963 . Since then, there was an experimental culture with juvenile yellowtail captured off the south coast . After the export of captured seeds for cultivating was banned in south korea in 1976, pond culture of yellowtail began in earnest . However, domestic aquaculture of seriola spp . Has so far been restricted to a method of pond culture of captured juveniles . In the future, culture production systems, such as seed production and intermediate rearing, clause 2 of the enforcement decree of the fishery resources management act (prohibition of capturing and gathering of fishery resources weight and length, presidential decree no . 25276), implemented from early 2014, it restricts the capture of yellowtail to individuals of 30 cm or shorter . In japan, the aquaculture of yellowtail kingfish used hormones for the induction of maturation and production of fertilized eggs (fugita & yogata, 1984). However, there has been no research into reproductive biology for the cultivation of yellowtail kingfish in south korea . This study therefore aimed to develop a method for the production of stable, healthy fertilized eggs from yellowtail kingfish . Since yellowtail kingfish are spring - summer spawning fish, that typically spawn april to june, broodstock management was conducted by controlling the light cycle in an indoor tank using the characteristics of spring - summer spawning, during which the spawning behavior, spawning induction, and spawning frequency were examined . In addition, the spawning amount, buoyancy rate and fertilization rate among spawned eggs, the size change of egg and oil droplet for buoyant eggs were investigated . Fifty - nine adult yellowtail kingfish were caught off the coast of jeju, south korea, and raised in a concrete indoor tank (8.0 8.0 2.0 m, usable capacity 100 m). In all, 20 fish (4.5 0.7 kg) were raised in march 2008, while the remaining 39 fish (2.6 0.7 kg) were raised in october 2012 (fig . The tank was filled with natural seawater, with the flow rate set at 6 cycles per day . The water temperature during the breeding period ranged from 12.0 to 29.0c . During the spawning season, the flow rate was adjusted to 3.5 cycles per day to protect the eggs, thus enabling good quality fertilized eggs to be collected . To induce maturation, a light intensity of 130 lux was maintained, in accordance with conditions for inducing maturation in spring - summer spawning fish (song, 2013), a light cycle of 12 h light (l): 12 h dark (d) was used from october 2013 to january 2014, and a light cycle of 15 h l: 9 h d from february 2014 to the end of spawning . Fish were fed a commercial ep feed (1618 mm), comprising 46.0% crude protein, 9.0% crude lipid, 16.0% crude ash, 5.0% crude fiber, 1.0% calcium, and 2.7% phosphorus . From april to the end of spawning, feeds were supplemented with pacific flying squid (todarodes pacificus). Rearing management for the induced sexual maturation of captive - reared yellowtail kingfish (s. lalandi) broodstock in indoor culture tank spawning began after sunset, between 19:00 and 24:00, and ended between 02:00 and 03:00 . Eggs were collected every morning between 08:00 and 09:00, before separating the buoyant eggs and sunken eggs using a measuring cylinder . The volume of each group was measured to calculate the spawning amount, while the buoyancy rate was calculated from the ratio of buoyant eggs to total spawning amount . The number of eggs was calculated volumetrically using a conversion rate of 500 eggs per ml . To calculate the rate of fertilization, a profile projector (pj300, mitutoyo) was used to count the ratio of buoyant eggs showing signs of development . The egg diameter and oil droplet diameter for 30 buoyant eggs were measured to a precision of 0.001 mm at 1-week intervals, using the profile projector . The growth of the broodstock in the concrete indoor tank during the breeding period is displayed in table 1 and fig . 2 . Those individuals acquired in march 2008, grew from 3.15.9 kg (mean 4.5 0.7 kg) to 9.414.9 kg (11.4 1.3 kg) in 77 months . Individuals acquired in october 2012 grew from 1.34.0 kg (2.6 0.9 kg) to 6.110.5 kg (8.0 1.1 kg) in 22 months . Males and females could not be distinguished owing to the similarity of their external appearance . Growth of captive - reared yellowtail kingfish (s. lalandi) broodstock used for the induction of sexual maturation and natural spawning in an indoor culture tank weight of captive - reared yellowtail kingfish (s. lalandi) broodstock used for the induction of sexual maturation and natural spawning in 2014 . 2008, wild - caught broodstock in 2008; 2012, wild - caught broodstock in 2012 . Following 3 months of long - day conditions (15l:9d), the spawning of yellowtail kingfish began in early may 2014, between the times of 19:00 and 24:00 . While the fish were swimming together in an orderly manner near the edges of the tank, a relatively large female was seen being followed by 2 males, twisting and intertwining . The males stuck close to the female s genital pore while swimming rapidly, and applied pressure to the female s abdomen . After approximately 1 hour of courtship behavior, the female began spawning, and the orderly swimming of males became started to spawn, in a frenzy of circling activity . After a while, the clear and still water in the tank became clouded with the spawned eggs and the sperm of the males, while small bubbles rose to the surface of the water . Spawning first began on may 3, when the water temperature was 17.0c, and it ended in mid - june, when the temperature was 20.5c . The yellowtail kingfish undergoing rearing management spawned a total of 26 times during the spawning period, with the total spawning amount being 4,449 10 individuals, making it an average spawning amount of 171 10 each time . Among mature yellowtail kingfish eggs, the mean ratio of buoyant eggs was 76.1%, while the fertilization rate for buoyant eggs during the spawning period was 100% (fig . 3). Right up until the third spawning event, the majority of eggs were cloudy or sunken, with a buoyancy rate of 0% . From may 23 to the end of spawning, the fish exhibited a pattern of spawning at 23 day intervals, with the buoyancy rate ranging from 33.396.4% . Spawn frequency and egg production of captivereared yellowtail kingfish (s. lalandi) broodstock in an indoor culture tank from may to june, 2014 . The egg diameter of fertilized yellowtail kingfish eggs was 1.388 0.041 mm, during which those eggs were buoyant with a single oil globule (fig . The egg diameter changed from 1.416 0.030 mm at the start of spawning on may 8, to 1.351 0.037 mm at the end of spawning on june 9 . Changes in mean egg diameter and oil globule diameter of natural spawned eggs from captivereared yellowtail kingfish (s. lalandi) broodstock in 2014 . The oil globule diameter at the start of spawning (may 8) was 0.348 0.025 mm, which gradually decreased towards the end of the spawning period . The egg diameter and oil globule diameter were reduced at the end of spawning by 4.1% and 13.0%, respectively, from that at the start of spawning . The growth of the broodstock in the concrete indoor tank during the breeding period is displayed in table 1 and fig . 2 . Those individuals acquired in march 2008, grew from 3.15.9 kg (mean 4.5 0.7 kg) to 9.414.9 kg (11.4 1.3 kg) in 77 months . Individuals acquired in october 2012 grew from 1.34.0 kg (2.6 0.9 kg) to 6.110.5 kg (8.0 1.1 kg) in 22 months . Males and females could not be distinguished owing to the similarity of their external appearance . Growth of captive - reared yellowtail kingfish (s. lalandi) broodstock used for the induction of sexual maturation and natural spawning in an indoor culture tank weight of captive - reared yellowtail kingfish (s. lalandi) broodstock used for the induction of sexual maturation and natural spawning in 2014 . 2008, wild - caught broodstock in 2008; 2012, wild - caught broodstock in 2012 . Following 3 months of long - day conditions (15l:9d), the spawning of yellowtail kingfish began in early may 2014, between the times of 19:00 and 24:00 . While the fish were swimming together in an orderly manner near the edges of the tank, a relatively large female was seen being followed by 2 males, twisting and intertwining . The males stuck close to the female s genital pore while swimming rapidly, and applied pressure to the female s abdomen . After approximately 1 hour of courtship behavior, the female began spawning, and the orderly swimming of males became started to spawn, in a frenzy of circling activity . After a while, the clear and still water in the tank became clouded with the spawned eggs and the sperm of the males, while small bubbles rose to the surface of the water . Spawning first began on may 3, when the water temperature was 17.0c, and it ended in mid - june, when the temperature was 20.5c . The yellowtail kingfish undergoing rearing management spawned a total of 26 times during the spawning period, with the total spawning amount being 4,449 10 individuals, making it an average spawning amount of 171 10 each time . Among mature yellowtail kingfish eggs, the mean ratio of buoyant eggs was 76.1%, while the fertilization rate for buoyant eggs during the spawning period was 100% (fig . 3). Right up until the third spawning event, the majority of eggs were cloudy or sunken, with a buoyancy rate of 0% . From may 23 to the end of spawning, the fish exhibited a pattern of spawning at 23 day intervals, with the buoyancy rate ranging from 33.396.4% . Spawn frequency and egg production of captivereared yellowtail kingfish (s. lalandi) broodstock in an indoor culture tank from may to june, 2014 . The egg diameter of fertilized yellowtail kingfish eggs was 1.388 0.041 mm, during which those eggs were buoyant with a single oil globule (fig . The egg diameter changed from 1.416 0.030 mm at the start of spawning on may 8, to 1.351 0.037 mm at the end of spawning on june 9 . Changes in mean egg diameter and oil globule diameter of natural spawned eggs from captivereared yellowtail kingfish (s. lalandi) broodstock in 2014 . The oil globule diameter at the start of spawning (may 8) was 0.348 0.025 mm, which gradually decreased towards the end of the spawning period . The egg diameter and oil globule diameter were reduced at the end of spawning by 4.1% and 13.0%, respectively, from that at the start of spawning . In broodstock management and rearing management for the induction of spawning in indoor tanks, it is important for the size and shape of the tank to be suited to the swimming characteristics and spawning behaviors of the broodstock . In previous studies, a 140-m circular tank was used for california yellowtail (seriola lalandi) (stuart and drawbridge, 2012), while spawning was induced in 14 yellowtail kingfish (mean weight, 17 kg) in a 70-m circular tank (moran et . In this study, the size of the tank was 100 m. the fish acquired in march 2008 grew from in mass from a mean of 4.5 0.7 kg to 11.4 1.3 kg in 77 months, while the fish acquired in october 2012 grew from 2.6 0.9 kg to 8.0 1.1 kg in 22 months . By adjusting the light cycle, we were able to induce spawning and retrieve fertilized eggs . In determining the size of broodstock management tanks for seriola spp ., it was assumed that there is some relationship between broodstock density and spawning behavior characteristics . Since this was the first time that fertilized eggs had been produced by yellowtail kingfish broodstock in an indoor tank, future studies will be required for the development of a breeding tank system that accounts for spawning behavior characteristics and the optimal male - to - female ratio and breeding density for stable healthy egg production . In new zealand yellowtail kingfish, spawning occurred 3090 minutes after courtship, but another male was observed to participate 1015 minutes prior to spawning (moranet al, 2007). In this study, we also observed 2 males entwining and twisting as they followed a single large female, sticking close to the genital pore, and swimming rapidly while pressing the abdomen of the female to induce spawning . Having information about the pre - spawning courtship behavior makes it possible to predict when spawning will occur and prepare for egg collection, thereby reducing the risk of bacterial infection due to delayed collection and mechanical damage from the collecting net (moran et al,2007). During this study, in which light cycle regulation was used to induce maturation of yellowtail kingfish in an indoor tank, it is difficult to find instances of broodstock management with light cycle - induced maturation of spring - summer spawning yellowtail kingfish off the coast of jeju; however, sevenband groupers (epinephelus septemfasciatus), which are summer spawning fish inhabiting the jeju region, undergo induction of maturation and fertilized egg production under similar broodstock management conditions of long days with water temperature regulation (song, 2013). Yellowtail kingfish fertilized egg production in goto in the south of japan was conducted with hormone treatment between april and may at a water temperature of 15.5 18.5c, followed by artificial insemination (fugita & yogata,1984). California yellowtail in california, san diego, and catalina island spawn between april and july at water temperatures of 16.522.0c, while yellowtail kingfish inhabiting east coast in northern new zealand spawn between november and february, when the water temperature is at least 17.0c (stuart & drawbridge, 2012; moranet al ., 2007). In terms of the size of individuals participating in spawning, one study of gonadal development in yellowtail kingfish reported that the minimum size fork length of females reaching maturity was 78 cm, 50% of females reached maturity at 94 cm, and 100% of females reached maturity at 128 cm ., 50% of males reached maturity at 81 cm, and 100% of males reached maturity at 93 cm (poortenaar etal ., 2001). Another study in new south wales, australia, showed that females first matured at 3 years and a fork length of 70 cm, and that 50% reached maturity at 83 cm between 45 years . In all, 50% of males matured at 47 cm and 1 year (gillanders etal . Mckenzie et al . (2014) reported that the reason why yellowtail kingfish living in new south wales and new zealand reach maturity and different sizes and ages is the water temperature, the unique habits of individuals, and the differences in the habitat conditions . In this study, the yellowtail kingfish broodstock were caught in the wild, and bred to a length of 85.0103.0 cm (mean 97.8 7.0 cm) in an indoor tank, meaning that, in contrast to the study of gonadal development in yellowtail kingfish off the coast of northern new zealand, the females should have exceeded the length at which they first started reaching maturity, while the males would have reached 100% maturity . A previous study showed that california yellowtails spawn 3743 times in water temperatures ranging from 16.522.0c (stuart & drawbridge, 2012). In the present study, this is consistent with results from southern japan, california, and new zealand, which showed that spawning begins at temperatures of 15c and above, but the regional differences in the number of spawns and the end of spawning can be considered to result from adaptive physiological characteristics . Fugita & yogata (1984) reported a fertilization rate of 81.6% for artificial insemination in yellowtail kingfish, while natural spawning by yellowtail kingfish in an indoor tank exhibited a fertilization rate of over 99% for buoyant eggs (moran et al ., 2007). In this study, the buoyancy rate was 76.1%, while the fertilization rate of buoyant eggs was 100% . Fugita & yogata (1984) measured the egg diameter and oil globule diameter of yellowtail kingfish that had been artificially inseminated following hormone treatment, and reported diameters of 1.36 0.07 mm and 0.34 0.02 mm, respectively . In this study, the egg diameter and oil globule diameter of yellowtail kingfish after natural spawning were 1.388 0.041 mm and 0.0378 0.029 mm, showing slightly greater size than that observed following artificial insemination . The egg diameter and oil globule diameter of california yellowtail were reported at 1.36 0.03 mm and 0.29 0.02 mm in 2009, and 1.40 0.05 mm and 0.32 0.05 mm in 2010 (stuart & drawbridge, 2012). Another study found that the egg diameter and oil globule diameter in yellowtail kingfish were 1.40 0.04 mm and 0.31 0.01 mm, respectively (moran et al ., 2007). The egg diameters in california yellowtail and yellowtail kingfish were similar . In this study, the egg diameter and oil globule diameter changed over during the course of spawning season, showing reductions of 4% and 13%, respectively, at the end of spawning from those at the start of spawning . The decrease in oil globule diameter size over the course of the spawning season also tended to follow the change in egg diameter . These results show that the egg diameter in yellowtail kingfish decreases volumetrically by approximately 15% during the spawning season, and that the oil globule diameter is closely related to the egg diameter and tends to decrease in size more than the egg diameter, resulting in a reduction in oil globule volume of approximately 20% from that at the start of spawning (moran et al ., 2007). The reasons for this reduction in egg diameter include differences between females in egg production and spawning period (fugita & yogata, 1984), as well as the general reduction in egg quality as the spawning period continues (brooks et al ., 1997). However, although egg quality is assumed to be better for larger eggs (kamler, 1992), there was no noticeable trend during the spawning period . It was therefore difficult to draw any conclusions about whether or not egg diameter affects egg quality (moran et al, 2007). Although this protocol produced fertilized eggs, future studies will be required investigating management to improve the health of broodstock for the stable acquisition of healthy eggs.
Cavernous sinus thrombosis (cst) was first reported by bright et al . In 18311 . Cst is an infectious disease that is potentially fatal if it is not treated in its early stage . Furthermore, despite treatment, complications of cst such as cranial nerve dysfunction may remain . There are very few reports of cst related to periodontal disease or to tooth extraction3 . The authors present a case in which misdiagnosis and inappropriate treatment of a buccal cheek laceration due to an ill - fitting denture resulted in a buccal space abscess that progressed into cst . A 65-year - old man with no past medical history presented to the emergency room with facial swelling and fever . Two weeks earlier, he began having trismus and pain on the mandibular left side . The patient was prescribed prednisolone, cyclobenzaprine, steroids and muscle relaxants and was advised to frequently apply moist hot packs . The patient's symptoms persisted and he developed spiking fevers with delirium . In the emergency room, his vital signs included blood pressure 151/82 mmhg, pulse 95 beats per minute, temperature 39.3, and respiratory rate 12/min . A complete blood count revealed a white blood cell (wbc) count of 19,110/l with 92.8% neutrophils, a erythrocyte sedimentation rate (esr) of 89 mm / h and c - reactive protein (crp) of 279.2 mg / l . Computed tomography (ct) showed an abscess in the buccal and pterygomandibular space spreading into the temporal space. (fig . 1) the patient's mental status was assessed with questions about his personal information . He had three teeth on the right mandibular side, and the left buccal cheek was swollen with a 1 cm laceration . However, because the patient showed clinical signs of infection associated with buccal, pterygomandibular, and temporal space abscesses, he was admitted to the hospital . Empirical antibiotic treatment was initiated with flomoxef 2 g / day, metronidazole 1.5 g / day, and isepamicin sulfate 400 mg / day . On the day of hospitalization ofloxacin otic solution was topically applied to the external auditory canal and the occlusive dressing was changed daily . On the second day of hospitalization, a spinal tap revealed cerebrospinal fluid turbidity suggestive of central nervous system (cns) infection . As per recommendations from internal medicine, and neurology, the antibiotic regimen was changed to ceftazidime 4 g / day and vancomycin 2.7 g / day to cover the cns infection and potential methicillin - resistance staphylococcus aureus (mrsa). On the third day of hospitalization, an oral and maxillofacial surgeon performed intraoral incision and drainage of the buccal and pterygomandibular spaces . Results of a magnetic resonance image (mri) included a dilated superior ophthalmic vein, bulging cavernous sinus contour, and septic thrombi in the cavernous sinus . 2) antimicrobial sensitivity testing identified methicillin - sensitive staphylococcus aureus (mssa) and therefore the antibiotic regimen was changed to nafcillin 12 g / day . His buccal swelling decreased, he recovered the ability to open his mouth, and his visual acuity and extremity sensation improved . Wbc, esr, and crp counts also improved. (table 1) with reduced swelling, we discovered an ill - fitting denture . Cst is a rare condition in which blood clots form within the cavernous sinus, a large collection of thin - walled veins . Septic cst may be caused by sinusitis, otitis, odontogenic infection, or facial cellulitis . The most frequent cause of septic cst is sinusitis in the sphenoid or ethmoidal sinuses . Dental infections account for <10% of septic cst cases, and most of these cases are related to maxillary infection4 . Eagleton5 proposed the following criteria for cst diagnosis: a known site of infection; blood stream infection (sepsis); early obstructive signs (such as retinal vein fullness, proptosis, exophthalmus, and collateral venous circulation); ocular nerve paralysis (via lesions of cranial nerves iii, iv, v, and iv); surrounding soft tissue abscesses (of the orbit, occiput, neck or nasopharynx); and symptoms of a complicated disease (such as headache, papilledema, and meningeal signs). Other potential pathogens include pneumococcus, bacteroides, fusobacterium, proteus, haemophilus, pseudomonas, and corynebacterium6 . Venous flow is bidirectional in the cerebral vein, emissary vein, and dural sinus because these vessels do not have valves . Therefore, bacteria or thrombi from another facial region can spread to the cavernous sinus via the facial vein or pterygoid plexus7 . Ophthalmoplegia is extraocular muscle weakness that results (in cst) when cranial nerves iii, iv, and/or vi are damaged due to their passage through the cavernous sinus . Other symptoms of lethargy, headache, periorbital swelling, papilledema, and venous engorgement occur in 50%-80% of patients . Decreased visual acuity, sluggish / dilated pupil, periorbital sensory loss, and decreased corneal reflex are less frequent symptoms that occur in <50% of patients . . Early symptoms of orbital cellulitis are similar to those of cst and include periorbital swelling, proptosis, chemosis, fever and impaired vision . Cst is included in the differential diagnosis when the above symptoms present bilaterally or when there is sensory loss in the periocular area, papilledema, and pupillary dilation9 . Coronal ct reveals that the lateral wall of the cavernous sinus is flat or convex instead of its normal concave shape . Additionally, irregular filling defects due to thrombi, and superior ophthalmic vein dilation are often observed . Mri is useful when the ct images are not clear or when the infection has spread to surrounding tissues including the brain and pituitary gland10,11 . In the past these imaging techniques are no longer recommended because they increase the risks of the infection spreading and thrombosis12 . Optimal therapy for cst includes the concomitant administration of nafcillin, metronidazole, and ceftriaxone / cefotaxime . Surgical interventions are indicated when the primary site of infection is sinusitis, or a dental infection . In the case of a serious infection, one may consider direct surgical drainage of the cavernous sinus; however, this approach is difficult and complications are likely to arise7 . Corticosteroid therapy must be considered if adrenal insufficiency occurs due to cranial nerve dysfunction or pituitary necrosis13 . Some studies have reported that anticoagulating with heparin, for example, within seven days after cst reduces the mortality rate14 . However, anticoagulation increases the risk of intracranial and/or systemic hemorrhage, and therefore should be used with caution15 . In this case, it was difficult to confirm the source of infection because the patient's buccal cheek swelling and trismus prevented adequate intraoral examination . The only suspicious finding was a small laceration on left buccal mucosa . Generally, odontogenic infections arise from the teeth themselves or from periodontal tissue . Therefore, it was difficult to confirm that this severe infection originated from a small laceration in a patient with no medical history . Another dentist who examined the patient two weeks before his presentation had misdiagnosed him with temporomandibular joint symptoms and myofascial pain . Administration of moist hot packs and prednisolone may have actually facilitated the infection's progression . Cst treatment must include proper management of the primary infection site and empiric antibiotics considering the possible pathogens . Cst presents with inconsistent symptoms, and therefore it is essential to collaborate with various departments including neurology, ophthalmology, otorhinolaryngology, and internal medicine . It presents with proptosis, chemosis, periorbital swelling and other typical signs of infection including as fever, pain and swelling . Clinicians in oral and maxillofacial surgery should be aware of cst and should consider it in patients with facial swelling.
During the last years an increased focus on fatigue symptoms / syndrome among children has been observed, together with an increase in referrals to our hospital . Children with fatigue whether these illnesses are increasing in number or just an increase in referrals to the specialist, in part based on greater interest in the topic is unknown . The aetiology of the condition is still unclear after almost three decades of research in this area, where infectious, immunological, neuroendocrine, sleep, and psychiatric mechanisms, among others, have been investigated . Despite the fact that no evidence has been found so far of an infectious aetiology, it has been proposed that a heterogeneous group of infections may trigger or maintain the symptoms of cfs . Abnormalities in the immune system have been reported in cfs - patients, among these, deficiencies in natural killer cell function and increased expression of activation markers on the cell - surface of t - lymphocytes, especially increased numbers of cd8 + cytotoxic t cells that have certain antigenic markers . Whether these abnormalities have any relationship to cfs and its symptoms remains unclear . The prevalence of the condition among young adults in the twenties and older has been reported as three percent studies have found a higher prevalence of cfs of two to three among females [1, 4, 8, 9]. Found that children and adolescents with chronic fatigue have a syndrome that is similar to that described in adults but that the syndrome presents earlier in the course of the illness and has a more optimistic outcome . A doctoral thesis from norway did not find socioeconomic status to contribute to the fatigue symptom, opposite to what chalder et al . Found . Having a disabling condition like cfs in childhood and adolescence may pose major consequences regarding mental health, personal relationships, school attendance and participating in social life and work . It is important to ensure early and proper management of these patients [15, 16]. The aim of the present hospital - based clinical retrospective study was (1) to estimate the number of children being diagnosed with g93.3 postviral fatigue syndrome during a 10-year period (2002 to 2011) and (2) describe the clinical and laboratory findings in children with postviral fatigue syndrome . The diagnosis given in icd-10 g93.3 postviral fatigue syndrome (cfs) is also used diagnosing children and adolescent with fatigue . However, time aspect with onset of symptoms is shorter for children than adults . All children and adolescents in the county where this study took place, was referred to specialist when diagnosing g93.3 post viral fatigue syndrome . All children suffering from fatigue are investigated in the department of paediatrics . During the first consultation the specialist will apply a preliminary diagnosis while the medical investigations are done . At the end of evaluation it should be noted that not all children with illness compatible with cfs will also fulfil criteria for postviral fatigue syndrome . The study was retrospective, and the subjects were all children under the age of 16 . A list of patients who underwent investigations and were diagnosed with g93.3 postviral fatigue syndrome according to the icd-10 classification was collected from the patient registry . The diagnosis was set by a paediatrician either during or at the end of the evaluation period . The following data were collected: (1) clinical examination, (2) blood sample tests, (3) other investigations, and (4) evaluation by physiotherapist, psychiatrist evaluating depression and anxiety using icd-10 criteria, and neuropsychologist assessing wisc - r measuring iq . Body mass index (bmi) was calculated retrospectively based on weight and length as kg / m . We defined overweight as bmi> 25 and underweight as bmi <17.5 . Because some findings support the notion that cfs may be due to one or more immune disorders that have resulted from exposure to an infectious agent, igg antibodies to epstein - barr virus (ebv) and cytomegalovirus (cmv) were used as a measure of previous infection . A total number of 37 patients were referred with fatigue symptoms according to the patient registry . Four patients were excluded due to obvious incorrect coding . Of the remaining 33, six (18%) two were diagnosed with epilepsy, two with sleeping disorders, one with hypothyreosis and one did not fulfil the criteria of cfs or any other diagnosis . Of the 27 who received the diagnosis g93.3, four had comorbid chronic illness . There were an increased number of referred patients with fatigue during the 10-year period, with almost three in four patients investigated during the last three years (20092011). Previous studies have found that many children with cfs do have mental health problems irrespective of cause [2023]. According to the psychiatrist assessment, none of the patients suffered from anxiety or depression . Three children had a pathological eeg; two of them had epilepsy and one sleeping disorder . Heart rate (hr) and blood pressure (bp) were all within normal range . One child had low ft4 and later diagnosed as hypothyreosis . Among the 27 patients referred with cfs, 16 (59%) were boys . The mean average age for the onset of fatigue symptoms was 141 months (sd 30) for boys and 136 months (sd 31) for girls, respectively . Being underweight, defined as bmi <17.5, was found in 12 (44%), while one (4%) was overweight with a bmi> 25 . Half of the underweight children were boys . Of the 27 patients with cfs (g93.3), 20 (74%) tested positive for igg to epstein - barr virus, six (22%) tested positive for igg to cytomegalovirus, and one (4%) tested positive to borrelia, indicating previous infection . In the present clinical retrospective study we found no gender differences among children and adolescent younger than 16 years of age with chronic fatigue symptom, with debut of symptoms after 10 years of age . Almost half of them were underweight and half of these were boys . Almost one in five suffering from fatigue symptoms proved to have other diagnoses . During the last decade, there have been an increase in number of referrals of children with fatigue as the only symptom . Chronic fatigue syndrome is not a new syndrome, there are documented cases since the 19th century . However during the last decade, there has been an increased knowledge and awareness about the condition in the healthcare system and in the media in norway which influence the number of referrals . In this study, the use of medical and biochemical test when assessing children and adolescence with chronic fatigue syndrome varied . The increased focus on the cfs diagnosis may have led to excessive use during the diagnostic evaluation process . The strength of the study is the retrospective design which allows us to confirm the diagnoses . In addition, due to uncertainty whether the whole county was covered, one cannot estimate incidences . Previous studies have reported a higher prevalence of cfs in the female population [25, 26]. However, this was a very small population, and it is possible that the results could have been different in a larger group . Time of onset seemed to be in accordance with other studies who describe symptom onset of fatigue between almost twelve until fifteen years . Almost all patients reported themselves to be previously healthy prior to their fatigue and also ascribed the onset due to an infection . One in six had a chronic condition that could explain their fatigue symptoms but still fulfilling criteria for g93.3 postviral fatigue syndrome . The diagnosis of cfs is usually established after excluding other chronic conditions; however, long standing medical or psychological conditions can also result in cfs . There is a gray area between fatigue due chronic medical and psychological conditions and fatigue which is a part of cfs . This zone needs to be examined carefully, and diagnostic criteria for cfs should be adjusted to clarify this overlap . Interestingly, we found that more than one in three of the referred patients was underweight, with an equal gender representation . The question is whether the underweight as a cause of fatigue has been overlooked in the diagnostic process or the cfs itself is the reason for underweight . Lack of the bmi records before the onset of fatigue symptoms makes it difficult to answer this question in our study . This is opposite to a recent study where high bmi was found to be significantly associated with prolonged duration of cfs in a retrospective cohort of children . Many of the adolescents had additional symptoms from the gastrointestinal system, like diarrhea and nausea [12, 28]. Further studies are needed to address the issue of eating disorders and to explain the reason for the low bmi . Many medical conditions present with long lasting fatigue as a disabling symptom [29, 30]. In this retrospective study, this is a reminder of the importance of a thorough investigation of the children being evaluated for possible cfs, to exclude other causes for their fatigue . The clinical assessment of children and adolescents with possible cfs needs systematic investigation . Nutritional status, possible eating disorder, and psychosocial issues need to be addressed and evaluated carefully.
Multiple sclerosis (ms) is a neurodegenerative disease characterized by chronic inflammation, demyelination, and scarring of the central nervous system . Symptoms include weakness, fatigue, sensory loss, vertigo, lack of coordination, impotence or sexual dysfunction, urinary incontinence, optic atrophy, dysarthria, and mental problems . The average age at onset of ms is 30 years, and the disease runs its course for the remainder of the patient's life frequently causing disability of varying degrees . The prevalence of ms varies with both geography and ethnic background with women twice as likely to be afflicted as men . With an estimated 35,000 sufferers, canada is considered a high frequency area with an average of between 55 and 202 per 10,000 persons . While the effect of ms on life expectancy remains controversial, the disease's negative effect on health - related quality of life (hrqol) is documented and a topic currently undergoing clinical study [5 - 7]. Several studies have shown that hrqol assessments provide unique information not measured by the kurtzke's expanded disability status scale (edss), the commonly used outcome measure of impairment disability for ms patients [8 - 10]. As an alternative indicators of the impact of the disease on a patient's life, self - reported hrqol focuses more attention on ms patients as a whole, in addition to focusing on physical problems . From the perspective of patients' organizations and regulatory agencies hrqol assessments are increasingly seen as additional sources of information on the safety and efficacy of treatments, particularly important in chronic progressive, disabling diseases for which there is no cure . Although the main use of hrqol instruments has been in the context of clinical trials, the observation that measures of patients' perception of their health do not overlap with clinician assessments of disability reinforces the importance of developing hrqol instruments for use in routine clinical practice . Rothwell et al . Have shown that physical disability may not always be the main determinant of overall health related quality of life . Furthermore, their study showed that patient with ms are less concerned than their clinicians about physical disability in their illness . Have shown that including hrqol in their multidimensional assessment protocol provides a detailed and sensitive evaluation of patients' disability profile and perceived difficulties thereby allowing clinicians to develop a care program tailored to each individual's needs . Hrqol measures can also serve as screening instruments for patients reporting changes in symptom severity or functional ability . The quality of life data can be used to involve patients and family members in clinical decision - making . Composed of the short form 36 item health survey (short form-36 or sf-36) and 18 disease specific items, this 54-item self - report measure combines the strength of generic and disease specific approaches to hrqol measurement . By complementing the sf-36 with questions focusing on the specific domains affected by ms it becomes possible to compare the hrqol of a patient suffering from ms with the hrqol of the general population and the hrqol of patients with other diseases . In routine clinical practice the instrument could be used to evaluate the differences in hrqol of individuals at various stages of ms . The msqol-54 has been used in the united states and has proven a valuable instrument for measuring hrqol in ms [21 - 23]. The questionnaire has also been translated into both italian and french . The objective of this study was to translate the msqol-54 into french canadian, and to make it available to the canadian scientific community for clinical research and clinical practice . According to the sapir - whorf hypothesis, every language embodies its own vision of the world, a prism through which its speakers inevitably view, for instance, their own health . We are in fact able to make the leaps of logic necessary to comprehend the complex ideas put forth by a foreign society, and translation techniques are available to remove the difficulties hindering the optimal transfer of the information, emotion, and stylistic content of the original message . Conducted using an internationally accepted methodology, the cultural adaptation of an hrqol questionnaire encompasses two essential stages: a translation stage ensuring linguistic validity of the questionnaire in the new language and the evaluation of the psychometric properties of the questionnaire or psychometric validation . The two are complementary and indispensable in demonstrating the equivalence between the original and the translated questionnaires . This paper describes the linguistic validation of the 18 ms specific questions (i.e. #37 to #54) in french canadian since the sf-36 has been previously translated, psychometrically validated and published in french canadian and is currently used in the canadian national population survey . More than one hundred million people of very diverse cultures speak french across the world . Quebec, the principal french - speaking center in north america, constitutes a significant member of this speech community . As is the case for all languages, there are occurrences of variation inside the french language . The variation is composite: it includes the pronunciation (the accents), sentence structure, and the lexicon, where the differences are most marked . For this reason we translated the 18 ms specific questions (i.e. #37 to #54) of the original us msqol-54 into french canadian instead of adapting the existing french version . In general, there are four major stages (figure 1) involved in a linguistic validation: forward translation, quality control through backward translation, test of understanding or cognitive debriefing, international harmonization . Each stage helps to improve the quality of the translation in terms of the conceptual equivalence between the original and translated questionnaires and the ease of understanding of the patient . In this project, the linguistic validation of the 18 disease - specific questions of the msqol-54 was performed solely into french canadian thereby removing the need for an international harmonization process . The linguistic validation was carried out in canada by a project manager based in canada, under the supervision of mapi research institute's central project manager based in lyon france . Mapi research institute process * * adapted from: mear i. difficulties of international clinical trials: cultural adaptation of quality of life questionnaires . Health - related quality of life and patient - reported outcomes: scientific and useful outcome criteria . The aim of this first step was to translate the 18 disease specific questions (#3754) of the original us msqol-54 questionnaire into french canadian and produce a version that was semantically and conceptually as close as possible to the original questionnaire . Two qualified translators, native speakers of french canadian, proficient in english and living in the target country, and the local french canadian project manager, performed this step . Each translator produced a forward translation of the original instrument into the target language without mutual consultation . The local project manager reviewed the forward translations, compared them with the original and established a consensus version . Carried out with quality control in mind, the 18 french canadian questions generated in step 1 were translated back into american english by one qualified translator, native speaker of english, proficient in french canadian and living in quebec . Following discussions between the translator, the local project manager, and mapi research institute's central project manager, translation discrepancies were corrected in the french version to generate a version of the msqol-54 ready for testing by ms patients . The aim of this step was to check the french canadian population's understanding and interpretation of the translated items and thereby validate the conceptual equivalence between the us and french canadian versions . The french canadian version of the questionnaire generated in step 2 was tested on five french canadian subjects with multiple sclerosis . According to the sapir - whorf hypothesis, every language embodies its own vision of the world, a prism through which its speakers inevitably view, for instance, their own health . We are in fact able to make the leaps of logic necessary to comprehend the complex ideas put forth by a foreign society, and translation techniques are available to remove the difficulties hindering the optimal transfer of the information, emotion, and stylistic content of the original message . Conducted using an internationally accepted methodology, the cultural adaptation of an hrqol questionnaire encompasses two essential stages: a translation stage ensuring linguistic validity of the questionnaire in the new language and the evaluation of the psychometric properties of the questionnaire or psychometric validation . The two are complementary and indispensable in demonstrating the equivalence between the original and the translated questionnaires . This paper describes the linguistic validation of the 18 ms specific questions (i.e. #37 to #54) in french canadian since the sf-36 has been previously translated, psychometrically validated and published in french canadian and is currently used in the canadian national population survey . More than one hundred million people of very diverse cultures speak french across the world . Quebec, the principal french - speaking center in north america, constitutes a significant member of this speech community . As is the case for all languages, there are occurrences of variation inside the french language . The variation is composite: it includes the pronunciation (the accents), sentence structure, and the lexicon, where the differences are most marked . For this reason we translated the 18 ms specific questions (i.e. #37 to #54) of the original us msqol-54 into french canadian instead of adapting the existing french version . In general, there are four major stages (figure 1) involved in a linguistic validation: forward translation, quality control through backward translation, test of understanding or cognitive debriefing, international harmonization . Each stage helps to improve the quality of the translation in terms of the conceptual equivalence between the original and translated questionnaires and the ease of understanding of the patient . In this project, the linguistic validation of the 18 disease - specific questions of the msqol-54 was performed solely into french canadian thereby removing the need for an international harmonization process . The linguistic validation was carried out in canada by a project manager based in canada, under the supervision of mapi research institute's central project manager based in lyon france . Linguistic validation mapi research institute process * * adapted from: mear i. difficulties of international clinical trials: cultural adaptation of quality of life questionnaires . In chassany o, caulin c, eds . Health - related quality of life and patient - reported outcomes: scientific and useful outcome criteria . The aim of this first step was to translate the 18 disease specific questions (#3754) of the original us msqol-54 questionnaire into french canadian and produce a version that was semantically and conceptually as close as possible to the original questionnaire . Two qualified translators, native speakers of french canadian, proficient in english and living in the target country, and the local french canadian project manager, performed this step . Each translator produced a forward translation of the original instrument into the target language without mutual consultation . The local project manager reviewed the forward translations, compared them with the original and established a consensus version . Carried out with quality control in mind, the 18 french canadian questions generated in step 1 were translated back into american english by one qualified translator, native speaker of english, proficient in french canadian and living in quebec . Following discussions between the translator, the local project manager, and mapi research institute's central project manager, translation discrepancies were corrected in the french version to generate a version of the msqol-54 ready for testing by ms patients . The aim of this step was to check the french canadian population's understanding and interpretation of the translated items and thereby validate the conceptual equivalence between the us and french canadian versions . The french canadian version of the questionnaire generated in step 2 was tested on five french canadian subjects with multiple sclerosis . The aim of this first step was to translate the 18 disease specific questions (#3754) of the original us msqol-54 questionnaire into french canadian and produce a version that was semantically and conceptually as close as possible to the original questionnaire . Two qualified translators, native speakers of french canadian, proficient in english and living in the target country, and the local french canadian project manager, performed this step . Each translator produced a forward translation of the original instrument into the target language without mutual consultation . The local project manager reviewed the forward translations, compared them with the original and established a consensus version . Carried out with quality control in mind, the 18 french canadian questions generated in step 1 were translated back into american english by one qualified translator, native speaker of english, proficient in french canadian and living in quebec . Following discussions between the translator, the local project manager, and mapi research institute's central project manager, translation discrepancies were corrected in the french version to generate a version of the msqol-54 ready for testing by ms patients . The aim of this step was to check the french canadian population's understanding and interpretation of the translated items and thereby validate the conceptual equivalence between the us and french canadian versions . The french canadian version of the questionnaire generated in step 2 was tested on five french canadian subjects with multiple sclerosis . As both gender populations will use these questionnaires both forms will be used in the translation wherever appropriate . It was suggested that " questionnaire de qualit de vie sur la sclrose en plaques " (quality of life questionnaire on multiple sclerosis) was not satisfactory as the notion of " sur la sclrose en plaques " (on multiple sclerosis) was not idiomatic . As a result of the discussions between the french canadian team and mapi research institute it was decided that " questionnaire de qualit de vie dans la sclrose en plaques"(quality of life questionnaire in multiple sclerosis) was more understandable and more accepted in spoken french canadian . First instruction sentence: " the survey asks you about your health and daily activities " in french canadian it is impossible to use a literal translation of the verb " to ask " in this context because in spoken french only a person can ask something of another person, a survey cannot . After discussion with the french canadian consultant, it was decided to render " asks you about " as " porte sur " (concerns), a much more idiomatic verb in this sense . Second instruction sentence: " answer every question by ticking the appropriate statement " as it is not idiomatic to use the verb " tick " in french canadian, it was decided to use " cocher " (cross), the usual way to mark answers in this language . Third instruction sentence: " if you are unsure how to answer a question, please give the best answer you can . " The notion " if you are unsure " was initially translated as " en cas d'hsitation " (in case of hesitation). However, subsequent to review and recommendation, it was decided to change this to " en cas de doute "(in case of doubt), as the french canadian population more readily understands it . Item 38: " were you discouraged by you health problems? " The item was originally translated as " avez - vous t dcourag(e) " (have you been discouraged). After review, it was recommended to harmonize the structure of this item with the one used in items 39 and 41 respectively in order to maintain fluency in french canadian . Thus " have you been discouraged " was changed to " vous tes - vous senti(e) dcourag(e) " (have you felt discouraged). This item was originally translated as " did your health worry you? " Which is not equivalent to the original as the notion of " in your life " was not included . Following the backward translation step it was decided to reword the question to include " dans votre vie " (in your life) to maintain the original documents intent and include a notion of duration . As a result " votre sant a - t - elle t un souci dans votre vie? " (has your health been a worry in your life?) Was adopted in french canadian . Item 43: " did you have trouble keeping attention on an activity for long " the original expression " keeping attention " was initially translated to " se concentrer " (to concentrate). This form was determined to be too restrictive since to concentrate is an action with no notion of duration . After discussions with the french canadian consultant following the backward translation step, it was decided to adopt " rester concentr(e) " (remain concentrated) to include the duration factor . Item 45: " have others, such as family members or friends, noticed that you have trouble with your memory or problems with your concentration? " The concept of " noticed " was initially translated as " pointed out to you " . During the backward translation step it was determined that the original did not reflect the notion that the family pointed this out to the patient and was worded in such a way that the patient might have heard these things indirectly or by chance . " Faire remarquer " (to point out) refers explicitly to a verbal exchange between the patient and his / her family or friends, while " remarquer " (remark, notice) simply refers to the fact that the patient's friends or family noticed his / her problems with memory but did not necessarily mention them to the patient . It was therefore determined to replace the concept of " faire remarquer " (pointed out) with " remarqu " (noticed) following the backward translation step . Subtitle: " sexual function " a literal translation was initially rendered but after cognitive debriefing the translation was deemed too technical . As a result item 49: " ability to satisfy sexual partner " the french canadian team initially suggested translating " ability " as " incapacity " given that items 46, 47, and 48 used negative wording to refer to sexual problems / dysfunctions . Item 46 refers to " lack of sexual interest ", 47 to " difficulty getting or keeping an erection / inadequate lubrication, and 48 " difficulty having orgasm " . Following discussion between the project manager and mapi research institute, it was determined that use of the term " incapacit " (incapacity) would carry a distinctly negative connotation thereby discouraging the patients to rate ability even if the ability exists in a reduced form . " Incapacit " was replaced by " capacit " (capacity) to better reflect the original document and thus remain more neutral, and less influential on patient's judgment of self . The french canadian version of the msqol-54 was tested on five french canadian subjects with multiple sclerosis . The length of the questionnaire was acceptable to the subjects with one exception who stated that the questionnaire should not exceed its current length as problems with concentration arose during the final phase of completion . No suggestions were made for missing articles or topics in the questionnaire.the translated msqol-54 went through cognitive debriefing successfully . As both gender populations will use these questionnaires both forms will be used in the translation wherever appropriate . It was suggested that " questionnaire de qualit de vie sur la sclrose en plaques " (quality of life questionnaire on multiple sclerosis) was not satisfactory as the notion of " sur la sclrose en plaques " (on multiple sclerosis) was not idiomatic . As a result of the discussions between the french canadian team and mapi research institute it was decided that " questionnaire de qualit de vie dans la sclrose en plaques"(quality of life questionnaire in multiple sclerosis) was more understandable and more accepted in spoken french canadian . First instruction sentence: " the survey asks you about your health and daily activities " in french canadian it is impossible to use a literal translation of the verb " to ask " in this context because in spoken french only a person can ask something of another person, a survey cannot . After discussion with the french canadian consultant, it was decided to render " asks you about " as " porte sur " (concerns), a much more idiomatic verb in this sense . Second instruction sentence: " answer every question by ticking the appropriate statement " as it is not idiomatic to use the verb " tick " in french canadian, it was decided to use " cocher " (cross), the usual way to mark answers in this language . Third instruction sentence: " if you are unsure how to answer a question, please give the best answer you can . " The notion " if you are unsure " was initially translated as " en cas d'hsitation " (in case of hesitation). However, subsequent to review and recommendation, it was decided to change this to " en cas de doute "(in case of doubt), as the french canadian population more readily understands it . Item 38: " were you discouraged by you health problems? " The item was originally translated as " avez - vous t dcourag(e) " (have you been discouraged). After review, it was recommended to harmonize the structure of this item with the one used in items 39 and 41 respectively in order to maintain fluency in french canadian . Thus " have you been discouraged " was changed to " vous tes - vous senti(e) dcourag(e) " (have you felt discouraged). This item was originally translated as " did your health worry you? " Which is not equivalent to the original as the notion of " in your life " was not included . Following the backward translation step it was decided to reword the question to include " dans votre vie " (in your life) to maintain the original documents intent and include a notion of duration . As a result (has your health been a worry in your life?) Was adopted in french canadian . Item 43: " did you have trouble keeping attention on an activity for long " the original expression " keeping attention " was initially translated to " se concentrer " (to concentrate). This form was determined to be too restrictive since to concentrate is an action with no notion of duration . After discussions with the french canadian consultant following the backward translation step, it was decided to adopt " rester concentr(e) " (remain concentrated) to include the duration factor . Item 45: " have others, such as family members or friends, noticed that you have trouble with your memory or problems with your concentration? " The concept of " noticed " was initially translated as " pointed out to you " . During the backward translation step it was determined that the original did not reflect the notion that the family pointed this out to the patient and was worded in such a way that the patient might have heard these things indirectly or by chance . " Faire remarquer " (to point out) refers explicitly to a verbal exchange between the patient and his / her family or friends, while " remarquer " (remark, notice) simply refers to the fact that the patient's friends or family noticed his / her problems with memory but did not necessarily mention them to the patient . It was therefore determined to replace the concept of " faire remarquer " (pointed out) with " remarqu " (noticed) following the backward translation step . Subtitle: " sexual function " a literal translation was initially rendered but after cognitive debriefing the translation was deemed too technical . As a result the french canadian term " item 49: " ability to satisfy sexual partner " the french canadian team initially suggested translating " ability " as " incapacity " given that items 46, 47, and 48 used negative wording to refer to sexual problems / dysfunctions . Item 46 refers to " lack of sexual interest ", 47 to " difficulty getting or keeping an erection / inadequate lubrication, and 48 " difficulty having orgasm " . Following discussion between the project manager and mapi research institute, it was determined that use of the term " incapacit " (incapacity) would carry a distinctly negative connotation thereby discouraging the patients to rate ability even if the ability exists in a reduced form . " Incapacit " was replaced by " capacit " (capacity) to better reflect the original document and thus remain more neutral, and less influential on patient's judgment of self . The french canadian version of the msqol-54 was tested on five french canadian subjects with multiple sclerosis . The length of the questionnaire was acceptable to the subjects with one exception who stated that the questionnaire should not exceed its current length as problems with concentration arose during the final phase of completion . No suggestions were made for missing articles or topics in the questionnaire.the translated msqol-54 went through cognitive debriefing successfully . Health - related quality of life (hrqol) questionnaires are increasingly used in international clinical trials . The main objective is to obtain a conceptual equivalence between the original and translated versions, allowing, among other things, a poolingand comparison of international studies data . This incremental methodological approach has become essential as increasing amounts of data are collected about cultural differences in measuring quality of life as well as the different types of equivalence between cultures . The translation of the msqol-54 in french canadian was carried out within the confines of internationally accepted methodologies under the supervision of experts in the field of cultural adaptation . As suggested by ware et al, hrqol questionnaires can form a practical tool for directly linking the norms from large population surveys with the results from more focused clinical trials, outcomes research studies, and monitoring efforts in everyday clinical practice . Patients have reported that information from assessments helped guide discussions about treatment options and care planning, thereby improving communication with health care providers . It has been suggested in the extent literature on doctor - patient relationships that the clinical application of hrqol instruments helps to open the lines of communication between doctors and patients . It signals to the patient that his or her doctor is prepared to discuss a wide range of health related issues thereby allowing them to relay details surrounding their condition that might otherwise remain unspoken . Of course, the use of hrqol questionnaires can never substitute for the natural dynamics of doctor - patient communication and interaction . It can, however, be viewed as a valuable tool for structuring the information gathering process . For these reasons, the french canadian msqol-54 can be considered as a useful tool to help health - care providers to conduct formal hrqol assessments of patients with ms as a routine part of clinical practice . Its proper use will necessitate the adherence to a few basic guidelines: 1) the average time of completion should not exceed 19 minutes, 2) the questionnaire may be administered directly in the waiting room, 3) the integrity of the instruments' wording should be maintained, 4) the patient's autonomy during completion process should be respected . The next step in the cultural adaptation process of the msqol-54 in french canadian will be field research to provide the empirical data necessary for its psychometric evaluation . The authors gratefully acknowledge the grant provided for this study by aventis pharma canada, and dr barbara vickrey for her support.
Insulin detemir has now become established as an effective basal insulin preparation and is widely used in the management of both type 1 and type 2 diabetes . Although it is similar in efficacy to isophane insulin, we have observed a relative lack of efficacy in two patients with significant hypertriglyceridemia and established nonalcoholic fatty liver disease, suggesting that there are circumstances where the efficacy of detemir may be adversely affected by specific metabolic parameters . Patient 1 was a 56yearold caucasian man with a 4year history of poorly controlled type 2 diabetes and marked hypertriglyceridemia (random triglycerides were 28.7 mmol / l and total cholesterol was 7.9 his mother had type 2 diabetes, but there was no other family history of note . He denied alcohol consumption for the previous 14 years and had been only a light drinker previously . At the time of starting insulin he was being treated with metformin, gliclazide, fenofibrate, and omacor . On examination, he was obese with a body weight of 109 kg and bmi of 32 kg / m . Glycated hemoglobin (hba1c) was 69 mmol / mol (8.5%), fasting triglycerides 8.4 mmol / l, and total cholesterol 4.5 mmol / l . Mol / l (egfr 102 ml / min/1.73 m) and liver enzymes were slightly elevated: ast 71 serum albumin was normal 47 g / dl (3550), inr 1.04, and hepatitis serology was negative . Ultrasound confirmed a 17.7cm spleen and ct demonstrated nonalcoholic fatty liver disease (nafld). In view of splenomegaly, he underwent a bone marrow aspirate and trephine biopsy, both of which were normal . A liver biopsy confirmed hepatosteatosis with marked steatosis, mild inflammatory activity, and hepatocellular ballooning (fig . 1a). In part of the biopsy, bridging fibrosis with parenchymal nodule formation was in keeping with cirrhotic transformation (fig . (b) reticulin stain (gordon and sweet) reveals, in part of the biopsy core, bridging fibrosis with parenchymal nodule formation in keeping with cirrhotic transformation . Insulin detemir (14 units nocte) was commenced as his random glucose values were 1722 mmol / l . He was using 158 units per day of detemir, in divided doses, plus insulin aspart 24 units with each meal . Despite these doses, mmol / l and hba1c remained unsatisfactory at 69 mmol / mol (8.5%). Basal insulin was then changed to insulatard 70 units per day in divided doses (insulin aspart dose unchanged) and within four months his hba1c had fallen to 51 mmol / mol (6.8%) and by 12 months he had lost 6 kg of weight . Patient 2 was a 68yearold man with a 20year history of type 2 diabetes and type v hyperlipidemia . For two years, after the diagnosis of diabetes, he was managed with oral hypoglycemic agents alone . He then developed necrotizing pancreatitis (considered secondary to hypertriglyceridemia) and was treated with human isophane insulin and human soluble insulin for ten years before being switched to detemir and novorapid . He was referred due to the patient's concern of escalating insulin dose and associated weight gain of 20 kg over the previous five years . At the time of referral the detemir dose was 120 units twice daily and novorapid 120 units with each meal (total of 600 units / day). He did not drink alcohol . On examination he was obese with body weight of 126 kg and bmi of 44.6 kg / m . Random triglycerides were markedly elevated (31.9 mmol / l), total cholesterol was 6.3 mmol / l, and hdl was 0.3 there was no clinical or biochemical evidence of any endocrinopathy to explain insulin resistance and no evidence of immunemediated insulin resistance . Liver enzymes and synthetic function were normal but there was mild renal impairment (serum creatinine 145 mol / l; egfr 77ml / min/1.73 m). Detemir was changed to isophane insulin 100 units bd and novorapid reduced to 100 units / meal . After six months, the dose of insulatard had stabilized at 140 units bd and novorapid 50 units tds (430 units / day 30% reduction in total daily dose), his weight remained unchanged and despite the reduction in insulin dose, hba1c improved to 50 mmol / mol (6.7%). The two cases presented in this report showed that in patients with a history of severe hypertriglyceridemia and significant fat infiltration of the liver, a hepatoselective insulin (detemir) appeared less efficacious in achieving glycemic control . Insulin detemir differs from human insulin by the deletion of the amino acid threonine in position 30 of the b chain, plus the addition of a c14 fatty acid chain at position 29 of the b chain . This allows detemir to reversibly bind to serum albumin 1 and is a characteristic that has led to it being considered a hepatoselective insulin: the albumin insulin molecule is unable to pass through the capillary endothelial cell barrier to reach peripheral adipocytes, whereas in the liver, the albumin detemir molecule is able to pass freely through the sinusoids . This allows it to exert a greater effect on hepatocytes than in peripheral tissues 2 . Hypothetically, the efficacy of hepatoselective insulin may be reduced in nafld by less hepatic exposure to insulin (due to increased insulin clearance or to portosystemic shunting), or from direct hepatic parenchymal cell damage . Given that the usual action of insulin is to stimulate the liver to store glucose in the form of glycogen and to switch off gluconeogenesis, underexposure of hepatocytes to insulin would lead to hyperglycemia . A portosystemic shunt was thought to be unlikely in these patients as there were no radiological features of collateral (variceal) circulation . Increased clearance of detemir might have occurred; premarketing studies suggested that hepatic impairment affected the bioavailability of insulin . In these studies there was a 3547% faster clearance of detemir in patients with severe / moderate liver insufficiency as compared to healthy subjects 3 . By contrast, clearance of human insulin is usually reduced in liver failure 4 and insulin clamp studies with human insulin have shown decreased clearance in patients with fat infiltration of the liver 5 . In a mouse model of nafld (induced by highfat feeding), there was decreased insulin activation of glycogen synthase and increased gluconeogenesis 6 during a euglycemic hyperinsulinemic clamp, suggesting a direct, adverse consequence of hepatic fat accumulation . The closest model available is of detemir's action in obesity where the presence of nafld may be inferred 7 . A pharmacodynamic study of 18 subjects showed that in the presence of grade 1 obesity (bmi 3035 kg / m) a lower glucose infusion rate was required to maintain normoglycemia after administration of detemir, compared to neutral protamine hagedorn (nph) insulin or glargine 8 . Advanced hepatocellular fibrosis may be associated with a change in the configuration of the sinusoidal endothelium, including loss of fenestrations, which would reduce the exchanges between hepatocytes and sinusoidal blood . Therefore, this would suggest that in severe nafld / cirrhosis, higher doses of detemir insulin would be required . An alternative hypothesis is that hypertriglyceridemia may have a direct, adverse effect on detemir action . The binding of detemir to albumin could be affected by the lipid content of plasma . Competition experiments with fatty acids have shown that levemir can be displaced from human albumin by longchain fatty acids (containing at least 12 carbon atoms) but with much weaker competition by a medium chain fatty acid 9 . The significance of this is uncertain, but potentially it could lead to loss of biological activity, or unpredictable release or clearance of levemir . Antibody development has been observed in phase 3 clinical trials of detemir, but did not impact on metabolic control 10 . There were no specific features of alternative cause(s) for a relative lack of efficacy of insulin, such as lysosomal storage disease or monogenic insulin resistance syndromes and these were not tested for . It is possible that the cases described relate to less common etiologies of insulin resistance . In managing patients with diabetes, interindividual differences in response to insulin may be encountered; a relative lack of efficacy with one type of insulin may not be seen with an alternative insulin . Such a response may be unrelated to the degree of hepatoselectivity of the insulin chosen . In conclusion, these two cases suggest that detemir may be less efficacious in patients with significant hypertriglyceridemia complicated by nafld . It is not possible to determine a precise mechanism to explain this observation but this could be due primarily to hypertriglyceridemia or due to the effects of infiltration of hepatic parenchyma with lipid.
Patients with coronary artery disease (cad) and diabetes have higher mortality and morbidity than patients without diabetes . Data from studies such as the uk prospective diabetes study suggest that very good glycemic control is associated with fewer cardiovascular events . Hypoglycemia is a very common side effect of insulin therapy and, to a lesser extent, of treatment with sulfonylureas . Risk factors for severe hypoglycemia include age, duration of diabetes, strict glycemic control, sleep, impaired awareness of hypoglycemia, renal impairment, c - peptide negativity and previous history of severe hypoglycemia . Acute hypoglycemia provokes pronounced physiological responses, the important consequences of which are to maintain the supply of glucose to brain and promote hepatic production of glucose . Hypoglycemia and the rapid changes in blood glucose have been shown to increase counter - regulatory hormones such as epinephrine and nor - epinephrine, which may induce vasoconstriction and platelet aggregation, thereby precipitating myocardial ischemia . Autonomic activation, principally of the sympatho - adrenal system, results in end - organ stimulation and the profuse release of epinephrine which precipitates hemodynamic changes like tachycardia, increased peripheral systolic blood pressure, decreased central blood pressure and increased myocardial contractility with an increased ejection fraction . The increased activity of sympathetic nervous system and secretion of other hormones and peptides such as the potent vasoconstrictor endothelin have pronounced effects on intravascular coagulability and viscosity . Increased plasma viscosity occurs during hypoglycemia because of an increase in erythrocyte concentration, while coagulation is promoted by platelet activation and an increment in factor viii and von - willebrand factor . Endothelial functions may be compromised during hypoglycemia because of an increase in c - reactive protein, mobilization and activation of neutrophils and platelet activation . The catecholamine - induced increased myocardial contractility may induce ischemia in the myocardium in patients with cad . The greater oxygen demand is not met because of not only the rigid vessels, but also endothelial dysfunction with failure to vasodilate . Several studies have shown that the hypoglycemia is associated with a significant lengthening of the corrected qt interval (qtc) in subjects with and without diabetes . These changes are likely seen because of increased catecholamine release during hypoglycemia, and qtc prolongation, in particular, could lead to a high risk of ventricular tachycardia and sudden death . Hyperinsulinemia and increased secretion of catecholamines may lead to hypokalemia during hypoglycemia, thus potentiating cardiac repolarizing abnormalities . Effects of antecedent hypoglycemia on cardiac autonomic regulation may contribute to the occurrence of adverse cardiac events . Abnormalities in high - frequency and low - frequency heart rate variability have been associated with hypoglycemia and increased catecholamine release . However, other studies did not find any associations between heart rate variability, hypoglycemia and increased catecholamine release . Episodes of hypoglycemia have been found to be associated with rise in inflammatory cytokines including interleukin (il)-6, il-8, tumor necrosis factor (tnf)-, c - reactive protein and endothelin-1 . These inflammatory cytokines result in endothelial injury and abnormalities in coagulation, resulting in rise of cardiovascular events . Inflammatory cytokines like il-1 have also been shown to increase the severity of hypoglycemia, thus perpetuating a positive feedback cycle . Vessel wall stiffness was found to be increased during hypoglycemia in patients with type-1 diabetes of longer duration than those with shorter duration of diabetes . Thus, hypoglycemia may increase the risk of cardiovascular events, especially in subsets of patients with longer duration of diabetes . Inflammation and endothelial dysfunction could potentially be the aggravating factors that contribute to increased cardiovascular risk with severe hypoglycemia, especially in the subset of patients with pre - existing cardiovascular disease, diabetes, and severe autonomic neuropathy . A direct relationship between hypoglycemia and fatal cardiovascular event is difficult to demonstrate as blood glucose and cardiac monitoring are seldom performed simultaneously . In the accord study, excess of deaths was noted in the intensive treatment arm, which led to discontinuation of study . In the smaller study of veterans with type-2 diabetes, veterans affairs diabetes trial (vadt), ecg changes, including ectopic activity, flattening of t - wave, st depression, ventricular tachycardia, and atrial fibrillation, have been reported in cases of low plasma glucose . Sudden death during sleep has been described in patients with type-1 diabetes, the mechanism being a significant cardiac arrhythmia induced by nocturnal hypoglycemia . Many of these patients have no evidence of severe hypoglycemia - induced neuronal damage at autopsy, implying that a cardiac arrhythmia had been triggered by hypoglycemia, resulting in sudden death . Despite the high frequency of nocturnal hypoglycemia in young patients with type-1 diabetes, sudden nocturnal death (dead in bed syndrome) is rare . Evidence is accumulating that severe hypoglycemia can provoke adverse cardiovascular outcomes such as myocardial ischemia or cardiac arrhythmia . Episodes of severe hypoglycemia are common during intensive therapy in type-1 and type-2 diabetes in the out - patient as well as in - patient setting . Larger clinical trials are required to look specifically at the association between hypoglycemia and cardiovascular events and to determine the mechanism further . The challenge to the physicians is to lower blood glucose to normal values to decrease the risk for long - term complications and at the same time minimize hypoglycemia and hypoglycemia - associated morbidity and mortality.
The extracellular matrix (ecm) is a protein rich entity which supports tissue structure, cell adhesion, cell - cell communication and differentiation 1 . Numerous studies have found that ecm proteins are aberrantly expressed in carcinomas and actively participate in tumor progression . In particular, fibronectin is receiving increasing interest as a result of its participation in multiple stages of tumor progression . Under homeostatic conditions, fibronectin has been shown to play a role in cell growth, differentiation, migration, and is additionally involved in processes such as wound healing and blood coagulation 2 . With regard to cancer, fibronectin is not only increased in tumors where its altered expression has been shown to promote tumor growth 3 - 5, migration 6, 7 and invasion 8, 9 but fibronectin has also been reported to limit tumor cell responsiveness to therapy 10 - 12 (figure 1). For instance, the introduction of fibronectin to differentiated mammary epithelial cell acini cultured atop the basement membrane extract matrigel caused the cells to re - enter the cell cycle thus reversing their growth arrest and acinar morphology 13 . Work in our lab has shown that loss of caveolin-1, the main structural protein of caveolae, results in increased expression of fibronectin, tenascin - c and collagen in murine mammary glands (thompson et al, unpublished data). The altered ecm is accompanied by changes in mammary gland architecture characterized by increased numbers of ducts that possess a larger circumference and area when compared to wild type controls, a result which may be in part related to increased fibronectin expression observed in the stroma (thompson et al, unpublished data). The purpose of this review is to illustrate the mechanisms in which aberrant fibronectin expression influences tumorigenesis and therapy resistance . In particular, this review will focus on the interactions between cell receptor ligands and fibronectin and how this interaction influences downstream signaling events that facilitate tumor progression . Further, this review will highlight potential strategies whereby fibronectin deposition, engagement with cell surface receptors and activation of cell signaling pathways may be exploited to halt tumor progression . In this manner, it is with anticipation that a new generation of novel therapeutics may be developed to better combat fibronectin's participation in tumorigenesis . Fibronectin has been shown to play a central role in processes associated with tumor progression . In particular, 51 integrin and fibronectin have not only been shown to be upregulated in tumors, but have also been reported to participate in tumor cell proliferation . For example, nam et al found that fibronectin and the extra domain (ed)-a splice variant of fibronectin were associated with higher 51-integrin expression in malignant as opposed to normal breast epithelial cells cultured atop matrigel 5 . Interestingly, the authors also reported that total fibronectin, ed - a fibronectin and 51-integrin was markedly upregulated in the malignant breast cancer lines but not in non - malignant breast epithelial cells 5 . Similarly, mierke et al also determined that 51 integrin was upregulated in mda - mb-231 breast cancer cells and further demonstrated that fibronectin augmented the invasiveness of 51 expressing breast cancer cells cultured atop collagen hydrogels 3 . To investigate a mechanism responsible for 51-fibronectin in tumor growth, mitra et al examined the activation of the receptor tyrosine kinase c - met in human heya8 and skov3ip ovarian cancer cell lines following 51 engagement with fibronectin 4 . Results showed that binding of cellular 51 to fibronectin resulted in increased activation of the c - met / fak / src signaling pathways in ovarian cancer cells 4 . Blocking ovarian cancer cell 51 interaction with fibronectin reduced c - met mediated focal adhesion kinase (fak) and src phosphorylation in vitro and in vivo and additionally reduced tumor weight and proliferation in xenograft tumors 4, suggesting that 51-fibronectin interactions regulate cell signaling pathways important for ovarian cancer growth . In an effort to further examine the effects of integrins on ovarian tumor cell proliferation, kenny et al examined adhesion and proliferation of human ovarian cancer cells following culture atop a de - cellularized ecm derived from human omental tumors 8 . When cultured in the presence of anti- 51, v3 and rgd peptides, the authors observed decreased adhesion and proliferation of ovarian cancer cells cultured atop the de - cellularized ecm supporting a role for fibronectin - integrin engagement in ovarian cell proliferation 8 . A defining feature of tumors is the ability of cancer cells to evade apoptosis . In an effort to delineate a role for fibronectin - integrin interactions on tumor cell resistance to apoptosis, han et al used wortmannin to block pi3-k in human bronchial epithelial beas-2b and 16-hbe cell lines following 51 cellular engagement with fibronectin 14 . The authors found that wortmannin caused an increase in dna fragmentation indicative of apoptosis 14 . In the absence of wortmannin, fibronectin stimulated 51-mediated phosphorylation of pi3-k and downstream signaling of pi3-k / akt, which in turn, down - regulated p21 and the tumor suppressor gene p53 and upregulated cyclin d1 in the bronchial epithelial cells 14 . In addition to integrins, fibronectin binding to cell surface receptors such as syndecans has been reported to regulate tumor cell ecm attachment and growth . This is evidenced by work from huang et al who examined the effect of tenascin - c on tumor cell adhesion to fibronectin 15 . Using mda - mb-435 breast cancer and t98 g gliobastoma cell lines, the authors found that tenascin - c bound to the fniii13 of the hepii site on fibronectin blocked syndecan-4 mediated tumor cell binding to fibronectin 15 . Overexpressing syndecan-4 reversed the effects of tenascin - c induced inhibition of tumor cell adhesion to fibronectin, suggesting that tenascin - c bound to the fniii13 site on fibronectin disrupts syndecan-4-fibronectin signaling 15 . Similarly, syndecan 1 and 4 engagement with fibronectin was reported to promote mda - mb-231 breast cancer cell adhesion and migration on collagen and proliferation on fibronectin matrices, an observation which was reversed upon incubation with anti - syndecan 1 and 4 antibodies 16 . Aside from direct cellular interactions with fibronectin on tumor progression, others have reported a role for fibronectin in tumor immunosuppression . For example, sengupta et al examined the role of sirna against fibronectin in glioma tumor growth and immune cell activation 17 . Using gl261 glioma tumor cells, the authors observed delayed tumor cell proliferation and growth in mice injected with fibronectin silenced gl261 cells 17 . This observation was in part, attributed to a reduction in the number of t - regulatory cells, which inhibit the activity of t helper cells . While the full length form of fibronectin plays an important role in tumorigenesis, isoforms of fibronectin, such as the ed - a and ed - b variants, have been reported to regulate tumor growth . For instance, rybak et al demonstrated markedly higher expression of ed - a fibronectin in the vasculature of murine liver metastases from f9 teratocarcinoma cells with minimal expression observed in non - diseased organs, pointing to a role for ed - a fibronectin in angiogenesis 18 . Indeed, a later study demonstrated that ed - a overexpressing colorectal cancer cells exhibited upregulated expression of the pro - angiogenic cytokine vascular endothelial growth factor - c (vegf - c) and promoted tumor growth and lymphangiogenesis in mice 19 . To determine a mechanism responsible for these observations, the authors report that ed - a upregulation of vegf - c is accomplished via ed - a mediated activation of the pi3k / akt pathway . Dose dependent reductions in vegf - c were observed following cancer cell treatment with an inhibitor of pi3k 19 . Aside from a role in angiogenesis, ed - a fibronectin was reported to be upregulated in patient colorectal tumors, especially those that were of advanced stage 20 . Investigating cd133/cd44 colorectal tumor cells, a subset of cells believed to be responsible for colon cancer initiation, the authors found that these cells expressed higher levels of the ed - a receptor 91 and further demonstrated that ed - a interaction with 91 was required for tumor cell proliferation 20 . To elucidate a mechanism responsible for their observations, ou et al found that ed - a sustains wnt/-catenin signaling, necessary for the progression of colorectal cancer cells, via integrin mediated activation of a fak / erk pathway 20 . In a more recent study, ed - a fibronectin was shown to increase the population of myeloid derived cells, known to impair immune responses to cancer cells, from osteoblasts 21 . To determine whether the absence of fibronectin impaired myeloid cell numbers and thus tumor formation, the authors subcutaneously injected melanoma cells into either a fibronectin knock out model (cko) in which fibronectin expression in differentiating osteoblasts was conditionally deleted or a wild type animal (wt) and evaluated for tumor formation 21 . Tumor growth and the presence of myeloid cells in tumors were reduced in cko animals but not in wt animals 21 . Although the authors don't attribute these findings to a specific reduction in the ed - a fibronectin isoform, they go on to demonstrate that culture of myeloid cells from cko animals with ed - a fibronectin resulted in a reduction of melanoma apoptosis, suggesting that this pro - tumor effect results from ed - a activation of myeloid cells 21 . Ed - b fibronectin has also been reported to play a role in angiogenesis where it has been shown to promote vegf expression, tube formation and proliferation of human vascular endothelial cells 22 . Specifically, the authors found that ed - b fibronectin was upregulated in vascular endothelial cells in response to high glucose, tgf-1 and endothelin-1, a finding which was reversed following administration of inhibitors to tgf-1 and endothelin-1 22 . These findings would suggest that growth factor signaling is an important regulator of ed - b fibronectin mediated vascular morphogenesis 22 . Despite a role in angiogenesis, ed - b fibronectin expression has been documented to be more highly expressed in the stromal and epithelial compartments of colorectal and breast carcinomas, but not in endothelial cells 23, suggesting that additional work is needed in order to fully elucidate the role of ed - b fibronectin in tumorigenesis . Regardless of its role in tumorigenesis, ed - b fibronectin has more recently been exploited as a molecular marker for prostate cancer . Here, han et al synthesized a small peptide to target upregulated ed - b expression in tumor xenografts harboring human prostate carcinomas, demonstrating that the peptide bound to ed - b fibronectin produced by the tumor cells 24 . It's conceivable that this strategy may be utilized for both imaging and diagnostic purposes as well as delivery of therapeutics . Overall, these studies suggest that fibronectin, via engagement with several integrin receptors and interference with immune function, favors tumor cell survival and proliferation . Additionally, multiple isoforms of fibronectin also participate in tumorigenesis, highlighting the importance of understanding how the different splice variants of fibronectin not only interact with cell surface receptors, but how this interaction activates downstream signaling pathways important for tumor growth . While fibronectin appears to play a key role in multiple facets of tumor progression, it has also been shown to participate in processes associated with tumor migration, invasion and metastasis . To examine the role of fibronectin on tumor cell migration, lou et al overexpressed sox2 to determine its role in ovarian tumor cell metastasis 6 . Here, the overexpression of sox2 upregulated fibronectin gene expression resulting in increased migration and invasion of the a2780 human ovarian cancer cell line in transwell chambers 6 . Downregulation of fibronectin using sirna resulted in a reversal of cell migration and invasion despite high expression of sox2 indicating that sox2 signals via fibronectin during tumor cell metastasis 6 . To further explore the role of fibronectin on tumor cell migration, wei et al sought to demonstrate whether nicotine enhanced the migration of sw480 colorectal cancer cells through nicotinic - acetylcholine receptor (7-nachr) mediated induction of cox-2 7, reported to regulate emt 25 . The authors found that inhibiting 7-nachr and cox-2 not only limited fibronectin expression, but also increased e - cadherin expression, reducing sw480 cell migration 7 . In the absence of cox-2 inhibition, nicotine enhanced colon cancer cell migration via 7-nachr and cox-2 mediated up - regulation of fibronectin expression indicating that inhibition of cox-2 and 7-nachr reduced migration of colorectal cancer cells as a result of a reduction in fibronectin expression 7 . Integrin engagement has been reported to play a crucial role in fibronectin mediated tumor cell invasion . A recent study reported an indispensable role for fibronectin and the 51 integrin on the invasive capabilities of heya8 and skov3ip1 human ovarian cancer cell lines 8 . Here, the authors first showed that 3d co - culture of ovarian cancer cells with mesothelial cells supported fibronectin expression from mesothelial cells via activation of a tgf-r1/rac1/smad3 signaling pathway, a phenomenon which was important for cancer cell adhesion, proliferation and invasion as silencing of fibronectin reduced these cellular responses 8 . In an attempt to further elucidate a role for fibronectin in tumor cell progression, the authors administered antibodies against 5 and 1 integrins to 3d co - cultures of ovarian cancer cells and mesothelial cells and discovered that tumor cell invasion and proliferation was reduced up to 40% 8 . In - vivo, the authors found that treatment of mice with antibodies against 5 and 1 reduced the number of metastases and tumor weight from orthotopically injected ovarian cancer cell lines 8 . These results suggest that ovarian cancer cells stimulate fibronectin expression from mesothelial cells via a tgf- pathway and that tumor cells adhere, proliferate and invade in response to 5 and 1 integrin engagement with fibronectin . In a similar study, it was shown that inhibition of 5 and 1 integrins significantly reduced ovarian tumor cell adhesion to a 3d model consisting of primary human mesothelial cells and fibroblasts and additionally limited the number of metastases in ovarian cancer xenografts 26 . Downregulation of e - cadherin on ovarian cancer cells was specifically shown to augment 5 integrin expression via activation of fak and erk1 26 . Further, sirna of e - cadherin was shown to increase cancer cell adhesion to fibronectin and invasion in a matrigel matrix 26, suggesting that ovarian cancer cell adhesion to fibronectin and invasion in a 3d matrix are regulated by decreased e - cadherin mediated 5 integrin expression . In addition to ovarian cancer, work by others have shown that mda - mb-231 breast cancer, t24 bladder carcinoma, and 786-o renal adenocarcinoma cells expressing 51 integrins exhibited increased invasion in 3d collagen matrices which had been polymerized with fibronectin, further highlighting that integrin engagement with fibronectin supports tumor cell invasion in 3d 3 . Interestingly, the addition of soluble fibronectin at the start of the assay reduced tumor cell invasion in collagen matrices, suggesting that differences in the polymerization status of fibronectin may differentially regulate tumor cell behavior 3 . A role for v3 interaction with fibronectin for example, 3d matrices of clotted plasma, reported to play an important role in tumor metastasis 27, or fibrin, the principal component of clots, were found to induce invadopodia, cellular protrusions which participate in the early stages of cell migration and invasion, formation from several tumor cell lines 9 . Furthermore, it was found that primary metastatic kidney tumor cells exhibited an increased expression of invadopodia formation in addition to fibronectin and v3 integrin which together upregulated the expression of the epithelial - mesenchymal (emt) transcription factor slug 9 . These results would suggest that v3 and fibronectin promote tumor cell invadopodia formation via activation of the emt transcription factor slug . In order to metastasize to distant sites, tumor cells make use of altered cell signaling pathways and matrix metalloproteinases (mmps), which degrade and remodel the ecm allowing tumor cell migration . With regard to a role for fibronectin in altered cell signaling, balanis et al showed that adhesion of mda - mb-231 breast cancer cells to fibronectin elicited robust stat3 activation, a result which was dependent on cellular expression of fak and the tyrosine kinase pyk2 28 . The authors further demonstrated that administration of fibronectin to mda - mb-231 cells cultured atop 3d hydrogels resulted in cellular outgrowth and a phenotype previously linked with enhanced metastatic properties 28 . Inhibition of 1 integrin or the use of a small inhibitor against stat3 reversed these findings, suggesting that in 3d, mda - mb-231 breast cancer cells utilize fibronectin-1 integrin mediated stat3 signaling during invasion 28 . To illustrate a cooperative role of fibronetin and mmps in tumor invasion, meng et al sought to determine whether a549 lung cancer cell invasion occurred as a result of fibronectin - induced fak activation of mmp9 29 . The authors found that fibronectin stimulated fak phosphorylation and src recruitment, resulting in activation of downstream targets, erk1/2 and pi3k / akt in a549 cells, promoting invasion and migration of the cancer cells in transwell chambers 29 . Inhibition of erk1/2 and pi3k resulted in a reduction of migrating and invasive a549 cells as a result of downregulation of mmp9, implicating a role for fibronectin - fak - mmp9 in lung cancer invasion 29 . Together, these findings not only suggest an important role for integrin engagement with fibronectin, but cooperation between fibronectin and altered cell signaling and mmp expression in processes associated with tumor invasion and metastasis . Chemotherapeutic agents have undoubtedly contributed to improved survival rates for individuals with cancer . Despite these successes, tumor cell resistance to therapies and relapse while a number of factors have been implicated in chemotherapy resistance, fibronectin has received interest . In a study by pontiggia et al the efficacy of tamoxifen, an estrogen receptor antagonist, was used to examine how the microenvironment limits tumor cell sensitivity to tamoxifen 12 . Here, the authors co - incubated human and mouse breast tumor cells with fibronectin and measured tumor cell sensitivity to tamoxifen 12 . The addition of fibronectin promoted tamoxifen resistance via fibronectin - mediated activation of 1-integrin which in turn, stimulated the pi3k / akt and mapk / erk 1/2 signaling pathways in both tumor cell lines 12 . Disrupting the interaction between the 1-integrin and fibronectin reversed the conferred resistance, leading to drug - induced apoptosis of the tumor cells 12 . Similarly, yuan et al found that 1 integrin and fibronectin were markedly increased in tamoxifen resistant as opposed to tamoxifen sensitive mcf7 breast cancer cells 30 . The overexpression of 1 was associated with increased expression of the epidermal growth factor receptor (egfr)/erk signaling pathway . Inhibition of 1 improved tamoxifen sensitivity of resistant mcf7 cells and further reduced cellular migration following culture in the presence of conditioned media from carcinoma associated fibroblasts (cafs) 30 . Interestingly, culture of tamoxifen resistant mcf7 cells in caf conditioned media resulted in enhanced cell migration due to caf produced fibronectin, a result which was diminished following cellular treatment with the 1 integrin inhibitor 30 . In addition to tamoxifen, cetuximab, an antibody that inhibits cell proliferation when bound to the egfr, has been reported to have limited efficacy as a result of tumor cell resistance 10 . For example, a549 human lung adenocarcinoma cells and h1299 human non - small cell lung carcinoma cells were reported to have diminished cell cytotoxicity to cetuximab and radiation following culture atop fibronectin coated surfaces 10 . The observed reduction of cytotoxicity was a result of tumor cell 51 integrin engagement with fibronectin as silencing these integrins restored tumor cell sensitivity to cetuximab 10 . Interestingly, cetuximab was also shown to promote increased fibronectin expression from both tumor cell lines, a finding which was reported to result from activation of the p38-mapk - atf2 signaling pathway 10 . Sirna silencing of fibronectin improved cytotoxicity of cetuximab in h1299 and a549 tumor cell lines, demonstrating that excess fibronectin in combination with 51 integrin engagement with fibronectin facilitates cetuximab resistance 10 . In a similar study, changes in tumor cell apoptosis were analyzed following etoposide treatment of h69 small cell lung cancer cells which had been cultured in fibronectin coated dishes 31 . The results showed that tumor cell 2, 3, 6, and 1 integrin interaction with fibronectin inhibited chemotherapy induced apoptosis, evidenced by decreased caspase 3-activity 31 . Using breast cancer as a model, spangenberg et al reported that inducible expression of the erbb2 proto - oncogene in mcf7 breast cancer cells resulted in upregulated gene expression of 5 and 1 integrins 32 . The authors found that mcf7 cells exhibited increased resistance to cisplatin and 5-fluorouracil as a result of erbb2 induced 51 integrin expression and exhibited a 2 fold reduction in responsiveness to these agents when the cells were cultured on fibronectin . To overcome chemotherapy resistance from integrin - fibronectin interactions, nam et al showed that disrupting the peptide bond between fibronectin and 51-integrin in combination with radiation therapy promoted apoptosis and reduced the expression of 51 in cultures of malignant human breast cancer cells cultured atop a 3d matrigel 5 . The authors found that the pro - survival effect of malignant tumor cell 51 interaction with fibronectin in a 3d environment was a result of 51 mediated activation of akt signaling as akt kinase and akt phosphorylation were downregulated following 51 inhibition 5 . Similarly, it was found that primary glioblastomas resistant to the anti - angiogenic agent bevacizumab exhibited a higher expression of 1 integrins and fak, a phenomenon the authors attributed to high intra - tumoral hypoxia following anti - angiogenic therapy 33 . To determine whether inhibition of 1 integrin improved therapeutic responses, the authors treated subcutaneously grown u87 mg glioblastomas with alternating doses of a 1 inhibitor and low dose (1mg / kg) bevacizumab 33 . The results showed a significant improvement in tumor regression in the dual treated animals 33 . These results were comparable to a high dose regimen (10mg / kg) of bevacizumab alone, suggesting that the addition of a 1 integrin inhibitor improves the therapeutic efficiency of bevacizumab 33 . Given these and other similar studies, it's apparent that targeting the cellular interactions with fibronectin and/or fibronectin itself may be a desirable strategy to improve patient outcomes . While the aforementioned studies have provided encouraging evidence to address therapeutic targets against integrin engagement with fibronectin, further research is necessary in order to find novel strategies for inhibiting tumor growth via targeting fibronectin biosynthesis and/or fibronectin regulated cell signaling . Matrix rigidity is a common feature of tumors and has been shown to support tumor growth, invasion and metastasis 34 . In response to matrix rigidity, focal adhesions, fak, a structural and signaling component of focal adhesions, has not only been reported to be upregulated in response to matrix rigidity 35, but has additionally been shown to regulate fibronectin - directed matrix fibrillar organization 36 . Ilic et al reported that fibronectin fibrils were more sparse and thinner in fak null embryos compared to wildtype littermates and fibroblasts, a result which was demonstrated to be independent of fibronectin gene or protein expression and synthesis, suggesting that fak activity is necessary for fibronectin matrix organization 36 . Interestingly, in a separate study, fibronectin expression was shown to drive fak signaling 29, suggesting that both fak and fibronectin may interact in a positive feedback loop . Given the role of fak in fibronectin expression, it's conceivable that targeting fak expression may be a mechanism to reduce fibronectin expression and thus tumor progression . Meng et al showed that fibronectin - induced fak tumor cell migration and invasion was decreased following treatment of a549 lung tumor cells with the src inhibitor pp2 29 . Specifically, the authors reported that inhibition of src resulted in decreased fak phosphorylation suggesting that a reduction of fak activity may be a useful strategy to reduce fibronectin mediated effects on tumor cell migration and invasion 29 . In addition to indirect targets of fak, it may be possible to directly reduce fak activity via use of a novel atp competitive kinase inhibitor, pf-04554878, which was recently shown to promote apoptosis and reduce proliferation of pancreatic neuroendocrine tumor cells 37 . Aside from targeting faks, it may be possible to directly reduce matrix stiffness in tumors, thereby preventing fak - induced fibronectin expression . Levental et al demonstrated that expression of lysyl oxidase (lox), an enzyme which cross - links collagen fibers 38, not only increased fak expression, but augmented matrix rigidity and breast tumor progression, a phenomenon which was reversed upon treatment with an inhibitor of lox 39 . In support of a role for lox in fibronectin expression, adam et al showed that atrial samples from patients with atrial fibrillation exhibited increased expression of lox and fibronectin 40 . Administration of a small molecule inhibitor of lox to neonatal cardiac fibroblasts attenuated lox - induced fibronectin expression via downregulation of angiotensin ii and connective tissue growth factor 40 . Although this work was conducted in cardiac fibroblasts, it's foreseeable that inhibiting lox may be a means to reduce tumoral fibronectin expression . Overall, it is likely that targeting the molecular machinery which is responsible for supporting matrix rigidity will reduce both fibrosis and fibronectin expression and in effect decrease tumor progression . While indirect targets against fibronectin should undoubtedly yield more favorable outcomes for cancer patients to this end, tomasini - johansson et al developed a small peptide inhibitor of fibronectin, pur4b, which was shown to limit fibronectin polymerization in the matrix 41 . Work by chiang et al has shown that administration of pur4b in animals which had received a partial ligation of the external or internal carotid arteries resulted in reduction of intima, media and adventitial thickness of the vessels and further decreased the accumulation of fibronectin and collagen 42 . Furthermore, hielscher et al demonstrated that treatment of co - cultures of fibroblasts and breast cancer cells with pur4b not only reduced fibronectin deposition and organization in the ecm, but also inhibited the deposition of other matrix proteins 43 . Additionally, inhibition of fibronectin matrix assembly also attenuated vascular morphogenesis of endothelial cells cultured atop the fibronectin - deficient matrix 43 . These studies illustrate that fibronectin not only serves as a substrate regulating the assembly of other ecm proteins but is additionally important for supporting angiogenesis . As such, direct inhibition of fibronectin polymerization via a small peptide may impair the assembly of other matrix proteins and capillaries, thus depriving tumors of a supportive scaffold and the oxygen and nutrients necessary for continued growth . Indeed, the use of such anti - cancer peptides have not only been used to directly treat cancer but have also been used as cytotoxic drug carriers 44 . As such, it's possible that delivery of a peptide inhibitor of fibronectin polymerization may be utilized in conjunction with a chemotherapy regimen to improve outcomes for cancer patients . A benefit from use of an inhibitor of fibronectin polymerization is that the off target effects of the peptide should be minimal as fibronectin isn't actively produced in tissues with the exception of wounds and during development . As such, one may need to complex the inhibitor in a delivery vector to improve both tumor targeting and sustained delivery of the peptide at the tumor site . Rna interference is rapidly becoming a powerful tool to silence the expression of genes and has been extensively investigated for its utility as a form of cancer therapy . Previous studies have shown that silencing fibronectin reduced tumor cell proliferation and growth 17 and tumor cell migration 6 . As such, silencing fibronectin gene expression via rna silencing may be an effective strategy to enhance therapeutic responses . Despite its obvious advantages, the major drawback associated with use of gene based therapies is the delivery of these to the tumor . Traditionally, sirna have been delivered locally where they've had much success with regard to treating ocular diseases 45 . For delivery of fibronectin sirna to tumors, it will be important to utilize vectors which not only target the tumor, but penetrate the tumor cell membrane to deliver the packaged sirna particles . Cell - penetrating peptides (cpp), which consist of short peptide sequences of 10 - 30 amino acids, have been shown to cross the cellular plasma membrane 46 . Derived from viral proteins or mammalian proteins with translocation capabilities, cpps have been used to deliver sirna to in - vitro cell cultures 47 and in transvascular delivery of sirna to mice 48 . Although additional work will be necessary, the use of these vectors may be an avenue by which to deliver fibronectin sirna to tumors . Another means by which to target fibronectin expression could come from use of vaccinia viruses, double stranded dna viruses used to treat cancer . Use of vaccinia viruses has received increased interest as strategies to engineer more potent oncolytic forms of vaccinia viruses has not only resulted in greater tumor specificity but have been shown to be an efficient means by which to lyse tumor cells 49 . Recently, a genetically engineered vaccinia virus was reported to target both malignant ovarian cancer cells in addition to the fibroblasts in the tumor stroma 50 . Given that the vaccinia virus was able to infect fibroblasts, it's possible that utilizing a similar strategy may halt the production of fibronectin from cancer associated fibroblasts as fibroblasts are a principle cell type responsible for ecm production . Together, the aforementioned studies have provided a clearer perspective on the mechanisms whereby fibronectin exerts its tumorigenic effects in cancer . These studies suggest that fibronectin is not only aberrantly expressed in tumors, but also promotes tumor progression, metastasis and therapy resistance via activation of a number of downstream signaling pathways . With advances in this knowledge, it's foreseeable that employing strategies to inhibit tumor cell association with fibronectin, fibronectin - mediated cell signaling and/or fibronectin biosynthesis in the ecm will not only result in unprecedented discoveries about the mechanisms fibronectin exerts on tumor cell behavior, but will additionally result in development of novel therapeutic approaches to treat cancer.
It is caused by fungi, which may be found in decaying food, in the soil, or other organic matter, such as animal excreta . Immunocompromised patients are especially susceptible, and timely diagnosis as well as intervention are of utmost importance for successful management . Because of atypical initial symptoms, such as facial pain, earache, sinus pain, or odontalgia, patients may seek dental treatment initially . Dental care may also precede such an infection, by means of a creation of a postextraction or postcurretage wound, which may be susceptible to fungal infection . The aim of this case report is to present a patient with rhinocerebral mucormycosis who was successfully treated due to timely diagnosis, effective surgical intervention, and intravenous antifungal treatment . A 22-year - old woman was transferred to the oral and maxillofacial department for facial pain, oedema, and double vision, following extraction of the upper right third molar tooth three days earlier . The patient had initially visited an ear - nose - throat specialist because of diffuse pain of the midface a week previously . The general dental practitioner had decided to extract the patient's upper right third molar tooth, which was in close contact to the inferior wall of the maxillary sinus, assuming that the symptoms were odontogenic . The patient had been hospitalized for generalized facial oedema and pain in a regional hospital but was transferred to our institution twenty - four hours later, when she developed double vision due to paralysis of the right lateral rectus muscle . The patient's medical history included diabetes mellitus type i, which was poorly controlled . On clinical examination, she presented slight hemifacial oedema of the right side, inward gaze of the right eye because of paralysis of the abduscent nerve (vi cranial nerve), as can be seen in figures 1 and 2 . Neurological examination also revealed hypaesthesia of the area of distribution of the maxillary nerve (2nd branch of the v cranial nerve). The right side of the palate was red with an ulcer of 1 cm diameter, near the second upper molar tooth (figure 3). Generally, the patient was an undernourished young woman, with normal vital signs, and unwilling to provide information regarding her diabetes or the reason for dental care . Undertaken investigations at admittance included blood count, serum electrolytes, urea, kreatinin, glucose, and c - reactive protein . Furthermore, glycated haemoglobin (glycohemoglobin, hba1c) was raised to 12.3%, with the normal range between 45.9% (table 1). Opthalmological examination showed palpebral oedema of the right side, no lesions of the retina or optic nerve, but diplopia at the right outward gaze, which confirmed the paralysis of the right lateral rectus muscle . Further imaging with magnetic resonance imaging also showed involvement of all paranasal sinuses of the right side, however, without involvement of the central nervous system (figure 4). A biopsy of the palatal ulcer showed fungal infection by species of mucorales (figure 5). The patient was initiated with intravenous antifungal agents (amphotericin b 300 mg qd and posaconazole 200 mg tid), and three days later she underwent subtotal right maxillectomy and reconstruction with an obturator . The paralysis of the right lateral rectus muscle progressively recovered, so the patient was dismissed from the hospital 2 months later, free of any infection, and has recently returned for definitive reconstruction (figure 6). During her hospitalization, our patient was also accustomed to more effective blood glucose management (table 1). The species of mucorales invade blood vessels and cause necrosis of vessel walls and mycotic thrombi . While healthy humans are resistant to such an infection, immunocompromised patients are generally much more vulnerable to the angioinvasive hyphal forms of these fungi . Diabetes mellitus, neutropenia, severe trauma, immunosuppression following transplantation of bone marrow, or solid organs are all predisposing factors for mucormycosis [1, 3, 4]. The host - specific condition may however render the host susceptible to different types of the infection . Mucormycosis is classified according to the anatomic site of occurrence in (1) rhinocerebral, (2) pulmonary, (3) cutaneous, (4) gastrointestinal, and (5) disseminated . Rhinocerebral or sino - orbital types are common among diabetic patients, especially those who are poorly controlled . The above - mentioned types of infection may present with various and atypical symptoms of sinusitis [1, 6]. Nasal congestion, headache, earache, or facial pain are some of the most common features, which are not at all characteristic . Depending on the affected site, adjacent structures like the orbit or the central nervous system may be involved . Similarly, extension to the cavernous sinus may cause cavernous sinus thrombosis [1, 2] or affect cranial nerves . Through the cribriform plate of the ethmoid bone or the supraorbital fissure, the infection may spread intracranially and cause abscesses or sagittal sinus thrombosis . The affected area is initially clear of any signs, but soon may appear reddish with or without necrotic eschars [1, 4]. Ct and mri are within normal limits initially, which are followed by signs of sinusitis, such as congested sinus or thickened mucosal lining . Repetition of the investigations is necessary for close followup of the advancement of the disease . A histopathological diagnosis is generally considered more precise than simple culture, as the latter may be unyielding due to the depth of invasion of the infection [1, 8, 9]. As soon as the clinical suspicion has risen, it is imperative to perform a biopsy of the area and initiate intravenous antifungal treatment [1, 2, 9]. Early intervention with ablative surgery is generally recommended [1014]. Especially considering rhinocerebral or rhino - orbital mucormycosis, enucleation of the eye although the internal medicine faculty strongly suggested total maxillectomy and removal of the eye, because of the aforementioned evidence, the absence of vision disturbance, severe conjunctival infection, or even optic nerve involvement in a young patient led to a more conservative maxillectomy . This fact is probably due to early intervention as well as no participation of the contents of the orbit, but cranial nerves . However, signs of involvement of the orbital area should always direct to a total maxillectomy, including the globe . The differential diagnosis of such a peculiar condition should initially include cocaine abuse, fasciitis, or other opportunistic infections of an immunocompromised host, such as aspergillosis, herpes simplex, or herpes zoster . Dental practitioners should be aware of rhinocerebral mucormycosis, specifically in cases of diabetic and other immunocompromised patients . Atypical symptoms such as facial pain, sinus pain, or unexpected odontalgia of otherwise healthy teeth should alert clinicians . Moreover, when a patient seems to deteriorate after dental therapeutic interventions, one should consider rare conditions, such as mucormycosis, and promptly urge the patient to seek medical advice . Particularly, meticulous intraoral examination should be performed to all patients, as atypical lesions may thus be revealed and estimated . In the case presented, a slight change of the mucosal colour or dehiscence of the palate might have been noticed . Similarly, it is imperative that all general dental practitioners note the patients' medical history and modify their diagnostic or therapeutic actions accordingly . As known, a raised glycated haemoglobin level is indicative of a poor blood glucose control by the diabetic patient . If the dentist had required a serum glucose or glycated haemoglobin test, he might have estimated the poorly controlled diabetic condition and might have avoided the unnecessary extraction of the upper right third moral tooth . Instead, a more precise dental examination might have shown the need for further investigation of the patient's facial pain.
Drug discovery is a complex and expensive endeavor that usually requires seven major steps: disease selection, target hypothesis, lead compound identification (screening), lead optimization, pre - clinical trial, clinical trial and pharmacogenomic optimization . Among the various techniques used to facilitate the drug discovery process, virtual or in silico ligand screening (vls) based on the structure of known ligands or on the structure of the receptor is becoming a method of choice (111), as seen in several recent studies [reviewed in (1216)]. It has been suggested that these libraries of purchasable small organic compounds should be filtered [adme / tox (absorption, distribution, metabolism, excretion and toxicity) filtering] in an attempt to work with databases of molecules with acceptable physical properties and chemical functionalities, at least consistent with known drug profiles (1727). Common filtering protocols can be variations of lipinski's rule - of - five (or ro5, potential for oral bioavailability) (25): molecular weight (mw) (poor absorption is observed if mw is more than 500), computed log p (p = octanol / water partition coefficient) (should not be more than 5), h - bond donors (should not be more than 5) and h - bond acceptors (should not be more than 10). Filters can also include a limit on the number of rotatable bonds, on the polar surface area (a value correlated to the number of h - bond donors and acceptors) among others, or can remove compounds containing specific chemical substructures associated with poor chemical stability or toxicity and sometimes attempt to predict drug metabolism (e.g. Cytochrome - mediated metabolism, pgp efflux) (2832). The selected molecules after applying lipinski's ro5 or related filters based on physicochemical properties or investigation of chemical functionalities are erroneously called drug - like while in fact, many organic compounds conform to the above listed rules but they are by no means drug - like (33). In fact, these rules define only some necessary conditions for a drug candidate (such as likely solubility, bio - availability) but not sufficient ones . Different levels of filtering could be applied in agreement with the aims of the project . For instance, soft filtering protocols are usually appropriate for cancer projects while, for some other studies, only small and rigid compounds / fragments (low mw, few rotatable bonds) are needed (e.g. Fragment - based lead discovery projects or fraganomics) (34). Only few online adme / tox tools are available, they can usually evaluate one compound at a time (table 1) while commercial packages are in general expensive [see review (35)]. Compound libraries can be found online, but they are usually not free nor filtered (table 2) (36). Only recently, a free 3d database of compounds ready for vls projects has been reported, zinc: (37). It is also possible to perform adme / tox computations via zinc and in this case they are carried out by the program filter (openeye scientific software, a program to remove undesirable molecules based on physicochemical properties and about 100 rules to eliminate unstable / reactive / dye chemical groups as well as to desalt the molecules). For the time being, the users of zinc can only apply default thresholds for the various computed properties . Because adme / tox calculations are usually not available online, we have created faf - drugs, a tool to perform physicochemical filtering . Also, in order to make vls experiments easier to perform to a broad community of users, we have interfaced several additional utilities (such as binding site prediction, openbabel) and processed five major compound collections . We use frowns (developed by brian kelley), a chemoinformatics toolkit () written in python and c++ to parse / read smiles (see explanations about the format at) or sdf files (see format at molecular design limited). We have implemented an algorithm in python that make use of frowns features to compute properties known to be important for filtering databases and that utilizes xtool (38) to compute log p - values . Because salts and counterions are often present in compound collections we recommend users to first apply the desalt utility that removes most salts and counterions prior to faf - drugs calculations . Then, our program computes the following molecular properties: (i) molecular weight (part of lipinski's ro5)(ii) hydrogen bond donors and acceptors (part of lipinski's ro5) (i) molecular weight (part of lipinski's ro5) (ii) hydrogen bond donors and acceptors (part of lipinski's ro5) defined as the number of hydrogen bond acceptors (sum of n + o) and hydrogen bond donors (sum of oh + nh). (iii) number of rigid bonds(iv) number of rings(v) size of the rings(vi) number of rotatable bond (iii) number of rigid bonds (v) size of the rings (vi) number of rotatable bond defined as any single non - ring bond, bounded to non - terminal heavy atom (29). The amide c - n bonds are not considered because of their high rotational energy barrier . (vii) number of carbon atoms, number of heteroatoms and ratio. (viii) number of atom with a net charge(ix) sum of formal charges(x) the topological polar surface area (tpsa) (vii) number of carbon atoms, number of heteroatoms and ratio . (viii) number of atom with a net charge (ix) sum of formal charges (x) the topological polar surface area (tpsa) the method described in (30) has been implemented . Briefly, the molecular polar surface area (psa) (i.e. Surface belonging to polar atoms) is a descriptor that was shown to correlate well with passive molecular transport through membranes . The calculation of psa, however, is rather time - consuming because of the necessity to generate a reasonable 3d molecular geometry and the calculation of the surface itself . A new approach for the calculation of the psa was developed by erlt et al . This approach was called topological polar surface area, it provides results that are practically identical with the 3d psa while the computation speed is 23 orders of magnitude faster . (xi) computation of xlogp (p = calculated octanol / water partition coefficient) (part of lipinski's ro5) (xi) computation of xlogp (p = calculated octanol / water partition coefficient) (part of lipinski's ro5) we use the xscore package () to compute xlogp as described in (38). This method gives log p - values by summing the contributions of component atoms while making use of correction factors . About 90 atom types are used to classify carbon, nitrogen, oxygen, sulfur, phosphorus and halogen atoms, and 10 correction factors are used for some special substructures . The contributions of each atom type and correction factor were derived by multivariate regression analysis of about 1850 organic compounds with known experimental log p - values . In faf - drugs, the format for the input files has, for the time being, to be sdf, smiles or cansmiles while the compounds have to be in mol2 format for xlogp computations . Few compounds are found to have ambiguous atom types and in this case the log p is not computed . (please see definitions about log p at:) (xii) atom check molecules with some specific atoms can be filtered - out (for instance molecules containing h, c, n, o, f, s, p, cl, br, i atoms are kept when using default parameters). We use frowns (developed by brian kelley), a chemoinformatics toolkit () written in python and c++ to parse / read smiles (see explanations about the format at) or sdf files (see format at molecular design limited). We have implemented an algorithm in python that make use of frowns features to compute properties known to be important for filtering databases and that utilizes xtool (38) to compute log p - values . Because salts and counterions are often present in compound collections we recommend users to first apply the desalt utility that removes most salts and counterions prior to faf - drugs calculations . Then, our program computes the following molecular properties: (i) molecular weight (part of lipinski's ro5)(ii) hydrogen bond donors and acceptors (part of lipinski's ro5) (i) molecular weight (part of lipinski's ro5) (ii) hydrogen bond donors and acceptors (part of lipinski's ro5) defined as the number of hydrogen bond acceptors (sum of n + o) and hydrogen bond donors (sum of oh + nh). (iii) number of rigid bonds(iv) number of rings(v) size of the rings(vi) number of rotatable bond (iii) number of rigid bonds (v) size of the rings (vi) number of rotatable bond defined as any single non - ring bond, bounded to non - terminal heavy atom (29). The amide c - n bonds are not considered because of their high rotational energy barrier . (vii) number of carbon atoms, number of heteroatoms and ratio. (viii) number of atom with a net charge(ix) sum of formal charges(x) the topological polar surface area (tpsa) (vii) number of carbon atoms, number of heteroatoms and ratio . (viii) number of atom with a net charge (ix) sum of formal charges (x) the topological polar surface area (tpsa) the method described in (30) has been implemented . Briefly, the molecular polar surface area (psa) (i.e. Surface belonging to polar atoms) is a descriptor that was shown to correlate well with passive molecular transport through membranes . The calculation of psa, however, is rather time - consuming because of the necessity to generate a reasonable 3d molecular geometry and the calculation of the surface itself . A new approach for the calculation of the psa was developed by erlt et al . This approach was called topological polar surface area, it provides results that are practically identical with the 3d psa while the computation speed is 23 orders of magnitude faster . (xi) computation of xlogp (p = calculated octanol / water partition coefficient) (part of lipinski's ro5) (xi) computation of xlogp (p = calculated octanol / water partition coefficient) (part of lipinski's ro5) we use the xscore package () to compute xlogp as described in (38). This method gives log p - values by summing the contributions of component atoms while making use of correction factors . About 90 atom types are used to classify carbon, nitrogen, oxygen, sulfur, phosphorus and halogen atoms, and 10 correction factors are used for some special substructures . The contributions of each atom type and correction factor were derived by multivariate regression analysis of about 1850 organic compounds with known experimental log p - values . In faf - drugs, the format for the input files has, for the time being, to be sdf, smiles or cansmiles while the compounds have to be in mol2 format for xlogp computations . Few compounds are found to have ambiguous atom types and in this case the log p is not computed . (please see definitions about log p at:) (xii) atom check molecules with some specific atoms can be filtered - out (for instance molecules containing h, c, n, o, f, s, p, cl, br, i atoms are kept when using default parameters). Online adme / tox tools are usually not freely available, for this reason, we have developed faf - drugs . This latter stands for free adme / tox filtering and drug - like compound collections . Our service can be used to filter collections available online as well as virtual libraries . Different levels of filtering have been reported in the literature, depending on the stage of the project, on the target and the disease types . For example, simple physicochemical property filtering could be used when searching for new hits on a new target while more complex adme / tox models (39) [see for example a list of chemical groups incompatible with final drug development (36,40)] could be applied at a later stage . We chose to implement only simple physicochemical rules because they address the filtering process using widely understood molecular properties . To start faf - drugs filtering, users can either write a molecule in smiles or 2d/3d sdf format directly in the web interface window or browse and upload a compound library . Salts and counterions are often present in compound collections and should be removed prior to adme / tox calculations . If salts and counterions are present, we suggest users to run first our desalt utility . At present, the input formats for faf - drugs calculations are cansmiles, smiles or sdf (please check our web site for explanations about the required formats) but openbabel (or online at rpbs) can be used for file format conversion prior to the filtering step (figure 1a). Then users can decide about the upper and lower limits of each investigated properties (adjustable thresholds) such as, to tailor the compound selection to a specific project . We also propose default parameters that are commonly used in the field (25,26,29,32,41). Users obtain two files with molecules that pass and do not pass the filters in cansmiles format together with the original (if available) compound i d provided by the chemical vendors . All computed properties (e.g. Mw, tpsa, xlogp) are also returned in a third file . In order to test our program, we performed computations on 50 080 molecules extracted from the chembridge compound collection (diversity set) with faf - drugs and filter (version 1.0.2, openeye scientific software) with the same parameters with the same threshold values (mw, tpsa). Both, filter and faf - drugs compute tpsa using the approach of erlt et al . A total of 49 334 passed the filters with faf - drugs and 49 032 with filter . Small differences could be due to the fact that some rules are implemented slightly differently, for instance tpsa or log p calculations or definition of flexible bond . Our tests on a linux machine (dell precision 650, 3ghz, 2 gb sdram) show that the standalone version of faf - drugs is able to process the above 50 080 molecules in about 20 min while equivalent computations on the same computer with filter (openeye) took about 10 min . Faf - drugs implementation is python - based and is not presently optimized for speed . With regard to server implementation, similar computations took about 30 min, but it can be longer (about 3 h) depending on the server load . We also compared faf - drugs with other online tools: molinspiration () that allows evaluation of few physicochemical properties (one molecule at a time can be processed, they have implemented their own tools to calculate log p while they follow the erlt et al . Approach to compute polar surface), and the log p calculators provided by syracuse research corporation (see table 1) and by tetko and tanchuck, alogps 2.1 (42). The method for log p prediction developed at molinspiration (milogp) is based on group contributions . These have been obtained by fitting calculated log p with experimental log p. alogps uses a neural network approach to predict logp while syracuse research corporation tool (logkow) estimates log p using an atom / fragment contribution method . Over 100 diverse molecules were tested and in all cases we computed very similar values . To illustrate our calculations, to further assess faf - drugs calculations, we compared over 100 computed log p - values [via our implementation of xlogp) with experimental log p (obtained via syracuse research corporation and via the edetox database ()]. The computed values are in good agreement with the experimental data, indicating that our implementation of xlogp is appropriate and that this approach gives very good results (figure 2). Taken together, the above data suggest that our adme / tox program is robust . Once users obtain the cansmiles output, they can decide about adjusting the filters and run additional computations or use 1d/2d to 3d conversion programs such as corina (), omega (openeye scientific software), converter (accelrys) and start a vls project . For the time being, to protect our server from intensive use, we suggest scientists to upload files with less than 30 000 molecules . In the present version of the service, computations for several tens of thousands of compounds remain time consuming (e.g. Several hours depending on the number of jobs in the queue) but work is in progress to improve this point . For this reason and in order to save cpu time and disk space, we also provide five filtered compound collections (figure 1a). A rational approach to increase the efficiency of finding new drugs and reduce the r&d cost is to reduce the attrition rate in the costly downstream stages (e.g. Clinical trials). Several important methods toward this goal have been developed, involving early computations of adme / tox properties . We have developed faf - drugs to help modelers and biologists to embark into drug discovery projects . Users can filter their own compound libraries and adapt the thresholds to a specific project . Other tools pertaining to the field of drug design / compound collections are also available at our web site . (a) schema of the faf - drugs service . Compound collections in smiles, cansmiles or sdf format are needed as input . Users obtain two output files, one with molecules that pass the filters and the other with compounds that do not pass the filters . A third file with all the computed properties several other utilities are available at faf - drugs, these involve online xlogp calculations (38) computed with xtool, online openbabel for file format conversion and implementation of the java molecular editor from dr p. ertl (novartis pharma ag, basel, switzerland) to draw molecules and obtain the corresponding smiles string . In addition, at faf - drugs, users can find five adme / tox filtered compound collections ready for vls computations . Three levels of filtering were applied (see our web site for further details) in order to better suit the needs of potential users . The openeye's omega program was used to generate 3d models, either single conformation or up to 50 conformations, for each molecule that passed the adme / tox filters . Other utilities consist of a test set that contains six protein targets (pdb format) and about 10 corresponding ligands (mol2 format, see information about the format at) to facilitate evaluation of docking / scoring methods and an interface to pass (43), a program that predicts binding pocket at the surface of a receptor . Many additional tools pertaining to the field of structural bioinformatics are also available at rpbs such as protein electrostatic computations, loop search, solvent accessibility prediction four molecules with different physicochemical properties were selected in order to compare faf - drugs calculations with other online tools . Example of free online adme / tox tools some online compound collections comparison of faf - drugs with several online tools faf - drugs computes several descriptors, such as molecular weight (mw), hydrogen bond donors (hd), hydrogen bond acceptors (ha), number of rotatable bonds (rot_bond), tpsa and log p (see text). Similar / identical results were obtained via the molinspiration website and by faf - drugs . Experimental log p - values and cas registry numbers were found in the edetox database () and at the (syracuse research corporation) server.

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